ABSTRACT
We have examined the respective influence of aldosterone, vasopressin and cell sodium delivery on Na+,K+-ATPase expression. The level of expression of the mRNA encoding for the alpha1- and beta1-subunits of Na+,K+-ATPase was evaluated in cortical collecting duct (CCD) cells from rats under different aldosterone status, in cells from the rat CCD cell line RCCD1 treated or not with vasopressin and in CCD cells from mice inactivated or not for the a-subunit of the epithelial sodium channel. The amount of mRNA was determined by in situ hybridization. Both aldosterone and vasopressin up-regulate transcripts encoding for the alpha1-subunit of Na+,K+-ATPase while beta1 is unaltered. Interestingly, when cell sodium entry was largely reduced (alphaENaC knock-out mice), the amount of transcripts encoding for the alpha1-subunit of Na+,K+-ATPase was significantly decreased in spite of high plasma aldosterone concentrations. No effect was observed on beta1-subunit. Altogether, these results suggest a coordinated hormonal and ionic control of Na+,K+-ATPase expression by different transcriptional pathways (steroid-receptor, cAMP-dependent and Na+dependent) in CCD cells. These regulations affect only alpha1-subunit of Na,K+-ATPase but not beta1.
Subject(s)
Aldosterone/pharmacology , Kidney Tubules, Collecting/enzymology , Sodium-Potassium-Exchanging ATPase/metabolism , Sodium/metabolism , Vasopressins/pharmacology , Adrenalectomy , Animals , Cell Line , Epithelial Sodium Channels , In Situ Hybridization , Kidney Cortex/drug effects , Kidney Cortex/enzymology , Kidney Tubules, Collecting/drug effects , Kidney Tubules, Collecting/physiology , Male , Mice , Mice, Knockout , Protein Subunits , RNA, Messenger/metabolism , Rats , Rats, Sprague-Dawley , Renal Agents/pharmacology , Sodium Channels/genetics , Sodium Channels/metabolism , Sodium-Potassium-Exchanging ATPase/geneticsABSTRACT
Several members of the H+,K+-ATPase family of ion pumps participate in renal K transport. This class of P-type ATPases includes the gastric H+,K+-ATPase as well as a number of nongastric H+,K+-ATPase isoforms. Physiological studies suggest that these enzymes operate predominantly at the apical surfaces of tubule epithelial cells. Although much has been learned about the pattern of H+,K+-ATPase isoform expression and its response to stress, the functional and cell biologic attributes of these pumps remain largely unelucidated. We have studied the properties of renal H+,K+-ATPases both in vitro and in situ. Our analysis of ion fluxes driven by a nongastric H+,K+-ATPase isoform suggests that it exchanges Na (rather than H) for K under normal circumstances. Thus, the individual H+,K+-ATPase isoforms may make diverse contributions to renal cation transport. We find that the activities of renal H+,K+-ATPases in situ are regulated by endocytosis, which is mediated by an endocytosis signal in the cytoplasmic tail of the gastric H+,K+-ATPase beta-subunit. Transgenic mice expressing a version of this protein in which the signal has been disabled show constitutively active renal K resorption. The identities of the H+,K+-ATPase isoforms that are normally subject to endocytic regulation and the nature of the participating epithelial cell machinery have yet to be established.
Subject(s)
H(+)-K(+)-Exchanging ATPase/metabolism , Kidney Tubules, Collecting/enzymology , Stomach/enzymology , Animals , Cells, Cultured , Humans , Ion Transport/physiology , Kidney Tubules, Collecting/metabolism , Mice , Mice, Transgenic , Urothelium/metabolism , Water-Electrolyte BalanceABSTRACT
Isoforms of the H-K-ATPase participate in active K resorption in the renal collecting tubule. The cytoplasmic tail of the beta-subunit of the gastric H-K-ATPase includes a 4 amino acid motif which is highly homologous to tyrosine-based endocytosis signals. We have generated transgenic mice expressing an H-K-ATPase beta-subunit in which the tyrosine residue in this sequence has been mutated to alanine. Mice expressing the mutated protein manifest constitutive hypersecretion of gastric acid, demonstrating that the beta-subunit tyrosine-based motif is required for the regulated endocytosis of the H-K pump and hence the cessation of gastric acid output. To test the possibility that the tyrosine-based sequence in the tail of the H-K-ATPase beta-subunit plays a role in regulating the function of renal H-K-ATPases, we examined renal K clearance in normal and in transgenic mice. Blood pressure, urine volume, glomerular filtration rate (GFR), plasma Na, and Na excretion are similar in control and transgenic mice. However, plasma K concentrations are significantly higher in transgenic mice (4.76 +/- 0.13 meq/l in transgenic and 4. 12 +/- 0.04 meq/l in control; n = 9, P < 0.05) and K excretion is lower in the transgenic animals (fractional excretion of K was 26.2 +/- 3.62% in transgenic and 50.1 +/- 4.78% in control; n = 9, P < 0. 01). These data suggest that the tyrosine-based signal in the cytoplasmic tail of the H-K-ATPase beta-subunit functions in the kidney as it does in the stomach to internalize H-K pump and thus inactivate pump function. Its elimination may result in the constitutive presence of the pump at the cell surface and lead to excessive urinary K reabsorption.
Subject(s)
H(+)-K(+)-Exchanging ATPase/metabolism , Kidney Tubules, Collecting/metabolism , Potassium/metabolism , Animals , H(+)-K(+)-Exchanging ATPase/chemistry , H(+)-K(+)-Exchanging ATPase/genetics , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Signal Transduction/genetics , Tyrosine/chemistry , Tyrosine/genetics , Tyrosine/metabolismABSTRACT
The Na,K-ATPase and the H,K-ATPase are highly homologous members of the P-type family of ion transporting ATPase. Despite their structural similarity, these two pumps are sorted to different destinations in polarized epithelial cells. While the Na,K-ATPase is restricted to the basolateral surfaces of most epithelial cells types, the H,K-ATPase is concentrated at the apical plasmalemma and in a pre-apical vesicular storage compartment in the parietal cells of the stomach. We have generated molecular chimeras composed of complementary portions of these two pumps' alpha-subunits. By expressing these pump constructs in polarized epithelial cells in culture, we have been able to identify sequence domains which participate in the targetting of the holoenzyme. We find that information embedded within the sequence of the fourth transmembrane domain of the H,K-ATPase is sufficient to account for this protein's apical localization. Stimulation of gastric acid secretion results in insertion of the intracellular H,K-ATPase pool into the apical plasma membrane and inactivation of acid secretion is accompanied by the re-internalization of these pumps. We have identified a tyrosine-based signal in the cytoplasmic tail of the H,K-ATPase beta-subunit which appears to be required for this endocytosis. We have mutated the critical tyrosine residue to alanine and expressed the altered protein in transgenic mice. The H,K-ATPase remains continuously at the apical cell surface in parietal cells from these animals, and they constitutively hypersecrete gastric acid. These results demonstrate that the beta-subunit sequence mediates the internalization of the H,K-ATPase and is required for the cessation of gastric acid secretion. Thus, at least two sorting signals are required to ensure the proper targetting and regulation of the gastric H,K pump.
Subject(s)
Cell Polarity/physiology , Epithelial Cells/enzymology , Epithelial Cells/physiology , H(+)-K(+)-Exchanging ATPase/metabolism , Sodium-Potassium-Exchanging ATPase/metabolism , AnimalsSubject(s)
Epithelial Cells/enzymology , H(+)-K(+)-Exchanging ATPase/chemistry , H(+)-K(+)-Exchanging ATPase/metabolism , Protein Structure, Secondary , Sodium-Potassium-Exchanging ATPase/chemistry , Sodium-Potassium-Exchanging ATPase/metabolism , Amino Acid Sequence , Cell Membrane/enzymology , Cell Polarity , Conserved Sequence , Epithelial Cells/physiology , Humans , Macromolecular Substances , Models, Molecular , Molecular Sequence Data , Receptors, Transferrin/chemistry , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Sequence Alignment , Sequence Homology, Amino AcidABSTRACT
Gastric acid secretion is mediated by the H/K-ATPase of parietal cells. Activation of acid secretion involves insertion of H/K-ATPase into the parietal cell plasmalemma, while its cessation is associated with reinternalization of the H/K-ATPase into an intracellular storage compartment. The cytoplasmic tail of the H/K-ATPase beta subunit includes a four residue sequence homologous to tyrosine-based endocytosis signals. We generated transgenic mice expressing H/K-ATPase beta subunit in which this motif's tyrosine residue is mutated to alanine. Gastric glands from animals expressing mutant beta subunit constitutively secrete acid and continuously express H/K-ATPase at their cell surfaces. Thus, the beta subunit's tyrosine-based signal is required for the internalization of H/K-ATPase and for the termination of acid secretion. As a consequence of chronic hyperacidity, the mice develop gastric ulcers and a hypertrophic gastropathy resembling Menetrier's disease.
