The molecular pathways for fluid transport in pulmonary, oral, and nasal tissues are still unresolved. Here we use immunocytochemistry and immunoelectron microscopy to define the sites of expression of four aquaporins in the respiratory tract and glandular epithelia, where they reside in distinct, nonoverlapping sites. Aquaporin-1 (AQP1) is present in apical and basolateral membranes of bronchial, tracheal, and nasopharyngeal vascular endothelium and fibroblasts. AQP5 is localized to the apical plasma membrane of type I pneumocytes and the apical plasma membranes of secretory epithelium in upper airway and salivary glands. In contrast, AQP3 is present in basal cells of tracheal and nasopharyngeal epithelium and is abundant in basolateral membranes of surface epithelial cells of nasal conchus. AQP4 resides in basolateral membranes of columnar cells of bronchial, tracheal, and nasopharyngeal epithelium; in nasal conchus AQP4 is restricted to basolateral membranes of a subset of intra- and subepithelial glands. These sites of expression suggest that transalveolar water movement, modulation of airway surface liquid, air humidification, and generation of nasopharyngeal secretions involve a coordinated network of aquaporin water channels.
Aquaporin (AQP) water channel proteins are tetrameric assemblies of individually active approximately 30 kDa subunits. AQP4 is the predominant water channel protein in brain, but immunoblotting of native tissues has previously yielded multiple poorly resolved bands. AQP4 is known to encode two distinct mRNAs with different translation initiating methionines, M1 or M23. Using SDS-PAGE urea gels and immunoblotting with anti-peptide antibodies, four polypeptides were identified in brain and multiple other rat tissues with the following levels of expression: 32 kDa > 34 kDa > 36 kDa > 38 kDa. The 34 and 38 kDa polypeptides react with an antibody specific for the N-terminus of the M1 isoform, and 32 and 36 kDa correspond to the shorter M23 isoform. Immunogold electron microscopic studies with rat cerebellum cryosections demonstrated that the 34 kDa polypeptide colocalizes in perivascular astrocyte endfeet where the 32 kDa polypeptide is abundantly expressed. Velocity sedimentation, cross-linking, and immunoprecipitation analyses of detergent-solubilized rat brain revealed that the 32 and 34 kDa polypeptides reside within heterotetramers. Immunoprecipitation of AQP4 expressed in Xenopus laevis oocytes demonstrated that heterotetramer formation reflects the relative expression levels of the 32 and 34 kDa polypeptides; however, tetramers containing different compositions of the two polypeptides exhibit similar water permeabilities. These studies demonstrate that AQP4 heterotetramers are formed from two overlapping polypeptides and indicate that the 22-amino acid sequence at the N-terminus of the 34 kDa polypeptide does not influence water permeability but may contribute to membrane trafficking or assembly of arrays.
Phosphorylation of Ser(256), in a PKA consensus site, in AQP2 (p-AQP2) appears to be critically involved in the vasopressin-induced trafficking of AQP2. In the present study, affinity-purified antibodies that selectively recognize AQP2 phosphorylated at Ser(256) were developed. These antibodies were used to determine 1) the subcellular localization of p-AQP2 in rat kidney and 2) changes in distribution and/or levels of p-AQP2 in response to [desamino-Cys(1),D-Arg(8)]vasopressin (DDAVP) treatment or V(2)-receptor blockade. Immunoelectron microscopy revealed that p-AQP2 was localized in both the apical plasma membrane and in intracellular vesicles of collecting duct principal cells. Treatment of rats with V(2)-receptor antagonist for 30 min resulted in almost complete disappearance of p-AQP2 labeling of the apical plasma membrane with only marginal labeling of intracellular vesicles remaining. Immunoblotting confirmed a marked decrease in p-AQP2 levels. In control Brattleboro rats (BB), lacking vasopressin secretion, p-AQP2 labeling was almost exclusively present in intracellular vesicles. Treatment of BB rats with DDAVP for 2 h induced a 10-fold increase in p-AQP2 labeling of the apical plasma membrane. The overall abundance of p-AQP2, however, was not increased, as determined both by immunoelectron microscopy and immunoblotting. Consistent with this, 2 h of DDAVP treatment of normal rats also resulted in unchanged p-AQP2 levels. Thus the results demonstrate that AQP2 phosphorylated in Ser(256) is present in the apical plasma membrane and in intracellular vesicles and that both the intracellular distribution/trafficking, as well as the abundance of p-AQP2, are regulated via V(2) receptors by altering phosphorylation and/or dephosphorylation of Ser(256) in AQP2.
Lithium (Li) treatment for 4 wk has previously been shown to increase the fraction of intercalated cells in parallel with a decrease in the fraction of principal cells in the kidney collecting duct (Christensen BM, Marples D, Kim YH, Wang W, Frøkiaer J, and Nielsen S. Am J Physiol Cell Physiol 286: C952-C964, 2004; Kim YH, Kwon TH, Christensen BM, Nielsen J, Wall SM, Madsen KM, Frøkiaer J, and Nielsen S. Am J Physiol Renal Physiol 285: F1244-F1257, 2003). To study how early this fractional change starts, the origin of the cells and the possible mechanism behind the changes, we did time course studies in rats subjected to different durations of Li treatment (i.e., for 4, 10, and 15 days). Increased urine output was already observed at day 4 of Li treatment with decreased AQP2 levels although not statistically significant. At days 10 and 15, both a significant polyuria and downregulation in AQP2 expression were observed. At day 10, the density of H+-ATPase-positive cells was increased in the IMCD of Li-treated rats and this was further pronounced at day 15. Some of the H+-ATPase-positive cells did not costain with Cl-/HCO3- exchanger AE1, indicating that they were not fully differentiated to type A IC. By double labeling for either H+-ATPase and proliferating-cell nuclear antigen (PCNA) or for AQP4 and PCNA, we found that proliferation mainly occurred in proximal IMCD cells at day 4 and it increased toward the middle part of the IMCD in response to prolonged Li treatment. Most cells expressing PCNA were stained with AQP4 but not with H+-ATPase. Triple-labeling for H+-ATPase, AQP4, and PCNA showed a subset of cells negative for all three proteins or only positive for PCNA. In contrast, a 4-wk recovery period after 4 wk of Li treatment reversed the enhanced proliferative rate to the control levels. In conclusion, the Li-induced increase in the density of intercalated cells is associated with a high proliferative rate of principal cells in the IM-1 and IM-2 rather than a selective proliferation of intercalated cells as expected. This is likely to contribute to the remodeling of the collecting duct after Li treatment.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.