Na+/K+-ATPase is a membrane protein and plays a key role in osmotic regulation in living organisms. In the present study, a cDNA sequence encoding the Na+/K+-ATPase alpha subunit from the monogonont rotifer, Brachionus koreanus was cloned by rapid amplification of cDNA ends technique. To investigate the role of this enzyme in osmotic stress, enzymatic activities of Na+/K+-ATPase were measured after exposure to different salinities for 48 h. The full-length Bk Na+/K+-ATPase cDNA was 3069 bp-long, encoding a 1022-amino acid polypeptide. Bk Na+/K+- ATPase possesses eight membrane spanning regions and five conserved domains. Phylogenetic analysis showed that Bk Na+/K+-ATPase had high identity with those of other species, and was closely clustered with other Brachionus sp. These findings indicate that this protein was conserved both structurally and functionally. B. koreanus Na+/K+-ATPase activity was stimulated in both hyposaline (6 psu) and hypersaline (32 psu) conditions, suggesting that this protein may play a role in osmoregulation. This study would provide better understanding of the physiology of B. koreanus and this enzyme may be useful as a molecular marker for evaluation of osmotic stress in aquatic environment.

Na+/K+-ATPase, an energy-transducing ion pump, is responsible for maintenance of relatively high concentrations of potassium ions but low concentrations of sodium ions in the cell by transport of these ions across the plasma membrane and participates in transport of various nutrients including glucose, amino acids. and ions. Na+/K+-ATPase consists of α, β, and FXYD subunits, but only α and β subunits are needed for basic functions. FXYD subunit is an auxiliary protein for αβ complex of Na+/K+-ATPase. Our recent study has shown that α (ATP1A1-4) and β (ATP1B1-3) subunits of Na+/K+-ATPase are expressed in the uterine endometrium during the estrous cycle and pregnancy in pigs. In this study, we further determined expression of FXYD (FXYD1-7) subunits of Na+/K+-ATPase in the uterine endometrium during the estrous cycle and pregnancy in pigs. Real-time RT-PCR analysis showed that mRNAs for all subtypes of FXYD subunit were expressed in the uterine endometrium during the estrous cycle and pregnancy in a pregnancy status- and stage-specific fashion. In situ hybridization analysis exhibited that transcripts of all subtypes of FXYD subunit were primarily localized to luminal (LE) and glandular epithelia (GE) during the estrous cycle and early pregnancy and to chorionic membrane (CM) during mid to term pregnancy. RT-PCR analysis showed that FXYD subunits were expressed in conceptuses on D12 and D15 of pregnancy. These results indicate that all subtypes of FXYD subunit are expressed in the uterine endometrium and conceptuses during the estrous cycle and pregnancy in a pregnancy status- and stagespecific manner. These suggest that FXYD may be involved in the establishment and maintenance of pregnancy by regulating the activity of Na+/K+-ATPase in nutrient transport at the maternal-fetal interface in pigs. * This work was supported by the Next Generation BioGreen 21 program (#PJ007997), RDA and the National Research Foundation (NRF #2010-0012304) funded by the Korean Government, Republic of Korea.

Na+/K+-ATPase, an energy-transducing ion pump, is responsible for maintenance of relatively high concentrations of potassium ions but low concentrations of sodium ions in the cell by transport of these ions across the plasma membrane. Na+/K+-ATPase consists of α, β, and γ subunits, but only α and β subunits are needed for basic functions. Na+/K+-ATPase is also involved in regulation of intracellular calcium ion concentration by coupling with Na+/Ca2+ exchanger involved in intracellular calcium extrusion. Our previous study showed that calcium regulatory molecules including Na+/Ca2+ exchanger are expressed in the uterine endometrium during the estrous cycle and pregnancy in pigs, however, expression of Na+/K+-ATPase in the uterine endometrium has not been determined. Thus, we examined expression of α1 (ATP1A1) and β1 (ATP1- B1) subunits of Na+/K+-ATPase in the uterine endometrium during the estrous cycle and pregnancy in pigs. Real-time RT-PCR analysis showed that levels of ATP1A1 m- RNA in the uterine endometrium during the estrous cycle and early pregnancy were higher than those during mid and term pregnancy, and that levels of ATP1B1 mRNA were highest on day (D) 12 of the estrous cycle. In situ hybridization analysis revealed that ATP1A1 and ATP1B1 mRNAs were localized to luminal (LE) and glandular epithelia (GE) in the endometrium. During mid to term pregnancy, localization of ATP1A1 mRNA was confined to LE, GE, and chorionic membrane (CM) of areolae and ATP1- B1 mRNA was localized to LE, GE and CM with the strongest intensity in LE of areolae. Signal intensity of ATP1B1 mRNA in LE was slightly stronger than that in GE. RT-PCR analysis showed that ATP1A1 and ATP1B1 mRNAs were expressed in conceptuses on D12 and D15 of pregnancy. These results showed that ATP1A1 and ATP1B1 were expressed in the uterine endometrium and conceptuses during the estrous cycle and pregnancy in a pregnancy status- and stage-specific manner. These suggest that Na+/K+-ATPase may play a key role in the establishment and maintenance of pregnancy by regulating intracellular concentrations of various ions including calcium at the maternal-fetal interface in pigs.

Na+/K+-ATPase, an energy-transducing ion pump, is responsible for maintenance of relatively high concentrations of potassium ions but low concentrations of sodium ions in the cell by transport of these ions across the plasma membrane and participates in transport of various nutrients including glucose, amino acids, and ions. In addition, Na+/K+-ATPase is also involved in regulation of intracellular calcium ion concentration by coupling with Na+/Ca+ exchanger expressed at the maternal-fetal interface in pigs. Na+/K+-ATPase consists of α, β, and FXYD subunits, but only α and β subunits are required for primary functions. FXYD subunit is an auxiliary protein for αβ complex of Na+/K+-ATPase. However, it has not been determined that subunits of Na+/K+-ATPase are expressed in the uterine endometrium during the estrous cycle and pregnancy in pigs. In this study, we determined expression of alpha (ATP1A1-4), beta (ATP1B1-3), and FXYD (FXYD1-7) subunits of Na+/K+-ATPase in the uterine endometrium during the estrous cycle and pregnancy in pigs. Real-time RT-PCR analysis showed that all alpha, beta, and FXYD subunits, except ATP1A3, were expressed in the uterine endometrium during the estrous cycle and pregnancy in a pregnancy status- and stage-specific fashion. In situ hybridization analysis exhibited that ATP1A1, ATP1A4, and ATP1B1 were localized to luminal (LE) and glandular epithelium (GE) during the estrous cycle and early pregnancy, and during mid to term pregnancy. ATP1A1 mRNA was localized to LE, GE, and areolae of the chorioallantois, especially at high levels to LE in areolae regions. ATP1B3 mRNA was detected only in LE during the estrous cycle and pregnancy with highest levels on day (D) 12 of pregnancy. Transcripts of all subtypes of FXYD subunit were primarily localized to LE and GE during the estrous cycle and early pregnancy and to chorionic membrane (CM) during mid to term pregnancy. RT-PCR analysis showed that all subtypes of Na+/K+-ATPase subunits, except ATP1A2, ATP1A3, and ATP1B2 mRNAs, were expressed in conceptuses on D12 and D15 of pregnancy. These results indicate that Na+/K+-ATPase subunits are expressed in the uterine endometrium and conceptuses during the estrous cycle and pregnancy in a pregnancy status- and stage-specific manner. These suggest that Na+/K+-ATPase subunits may be involved in the establishment and maintenance of pregnancy by coordinate regulation of absorption and secretion of nutrients such as glucose, amino acids, and ions at the maternal-fetal interface in pigs.