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This study sought to establish the presence of K(+)-activated adenosinetriphosphatase (ATPase) activity in the colonic mucosa of the rat distal colon. K(+)-activated ATPase activity was present in apical membranes but not in basolateral membranes. K(+)-activated ATPase activity in apical membranes represented an approximate 10-fold enrichment compared with that in the homogenate. Na(+)-K(+)-activated ATPase activity was also present in homogenate but was enriched less than fourfold in apical membranes. K(+)-activated ATPase activity in apical membranes had both ouabain-sensitive and ouabain-insensitive components. In contrast, Na(+)-K(+)-activated ATPase activity was completely inhibited by ouabain. Similar half-maximal concentrations for K+ and pH activation curves were found for both ouabain-sensitive and ouabain-insensitive fractions. In addition to K+, the ouabain-sensitive fraction of K(+)-activated ATPase activity was stimulated by Rb+, NH+4, and Cs+, whereas the ouabain-insensitive fraction was activated only by Rb+. K(+)-activated ATPase activity was significantly inhibited by vanadate but not by N-ethylmaleimide or omeprazole. In the proximal colon, in contrast to the distal colon, active K+ absorption is not present, and K(+)-activated ATPase is approximately 20% of that in the distal colon. These studies demonstrate that K(+)-activated ATPase is present in apical membranes of rat distal colon and permit the speculation that this enzyme represents a unique and distinct ATPase (compared with either Na(+)-K(+)-ATPase or gastric parietal cell K(+)-ATPase) and is likely linked closely to the active K+ absorptive process present in this epithelium.

Transepithelial Na+ transport is mediated by passive Na+ entry across the luminal membrane and exit through the basolateral membrane by two active mechanisms: the Na+/K+ pump and the second sodium pump. These processes are associated with the ouabain-sensitive Na+/K+-ATPase and the ouabain-insensitive, furosemide-inhibitable Na+-ATPase, respectively. Over the last 40 years, the second sodium pump has not been successfully associated with any particular membrane protein. Recently, however, purification and cloning of intestinal α-subunit of the Na+-ATPase from guinea pig allowed us to define it as a unique biochemical and molecular entity. The Na+- and Na+/K+-ATPase genes are at the same locus, atp1a1, but have independent promoters and some different exons. Herein, we spotlight the functional characteristics of the second sodium pump, and the associated Na+-ATPase, in the context of its role in transepithelial transport and its response to a variety of physiological and pathophysiological conditions. Identification of the Na+-ATPase gene (atna) allowed us, using a bioinformatics approach, to explore the tertiary structure of the protein in relation to other P-type ATPases and to predict regulatory sites in the promoter region. Potential regulatory sites linked to inflammation and cellular stress were identified in the atna gene. In addition, a human atna ortholog was recognized. Finally, experimental data obtained using spontaneously hypertensive rats suggest that the Na+-ATPase could play a role in the pathogenesis of essential hypertension. Thus, the participation of the second sodium pump in transepithelial Na+ transport and cellular Na+ homeostasis leads us to reconsider its role in health and disease.

Primary Na+ transport has been essentially attributed to Na+/K+ pump. However, there are functional and biochemical evidences that suggest the existence of a K+-independent, ouabain-insensitive Na+ pump, associated to a Na+-ATPase with similar characteristics, located at basolateral plasma membrane of epithelial cells. Herein, membrane protein complex associated with this Na+-ATPase was identified. Basolateral membranes from guinea-pig enterocytes were solubilized with polyoxyethylene-9-lauryl ether and Na+-ATPase was purified by concanavalin A affinity and ion exchange chromatographies. Purified enzyme preserves its native biochemical characteristics: Mg2+ dependence, specific Na+ stimulation, K+ independence, ouabain insensitivity and inhibition by furosemide (IC50: 0.5 mM) and vanadate (IC50: 9.1 μM). IgY antibodies against purified Na+-ATPase did not recognize Na+/K+-ATPase and vice versa. Analysis of purified Na+-ATPase by SDS-PAGE and 2D-electrophoresis showed that is constituted by two subunits: 90 (α) and 50 (β) kDa. Tandem mass spectrometry of α-subunit identified three peptides, also present in most Na+/K+-ATPase isoforms, which were used to design primers for cloning both ATPases by PCR from guinea-pig intestinal epithelial cells. A cDNA fragment of 1148 bp (atna) was cloned, in addition to Na+/K+-ATPase α1-isoform cDNA (1283 bp). In MDCK cells, which constitutively express Na+-ATPase, silencing of atna mRNA specifically suppressed Na+-ATPase α-subunit and ouabain-insensitive Na+-ATPase activity, demonstrating that atna transcript is linked to this enzyme. Guinea-pig atna mRNA sequence (2787 bp) was completed using RLM-RACE. It encodes a protein of 811 amino acids (88.9 kDa) with the nine structural motifs of P-type ATPases. It has 64% identity and 72% homology with guinea-pig Na+/K+-ATPase α1-isoform. These structural and biochemical evidences identify the K+-independent, ouabain-insensitive Na+-ATPase as a unique P-type ATPase.

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