This proposal continues studies that use combined physiological and ultrastructural analysis to develop a detailed understanding of vasopressin action in the toad urinary bladder. The following new strategies developed in the last grant period will be used to elucidate the role of membrane structural features in the water permeability response: 1) """"""""Sandwich"""""""" replica analysis combines features of freeze-fracture and deep-etching to provide a unique view of the interrelationship between organized elements on the surface of membranes and intramembrane structure 2) """"""""Hyperstretch"""""""" of bladders incubated in ice-cold Ringer flattens the apical membrane nearly completely to allow a comprehensive evaluation of the incidence and area of intramembrane particle aggregates in vasopressin-treated bladders 3) """"""""Label-fracture"""""""" analysis of extracellular markers endocytosed with removal of vasopressin demonstrates that intact aggregates are retrieved from the apical surface with conditions that reduce the apical membrane permeability response. Novel applications of this method now allow two types of aggregate containing membrane vesicles to be distinguished in the cytoplasm following vasopressin removal and permit a quantitative evaluation of the role of membrane retrieval in regulation of the permeability response. In addition, methods that allow selective labeling of retrieved membrane constituents have led to a promising new strategy for isolation of these structures and the identification of aggrephore-specific proteins. Using antibodies to apical membrane proteins we will evaluate the specificity of membrane retrieval responses. We will use monoclonal antibodies to aggrephore components and label-fracture methods to localize these components with respect to the intramembrane particle aggregates. This strategy should allow us to distinguish which protein(s) of the complex aggrephore vesicles directly relates to the aggregates and therefore to the channels of interest. In addition, labeling studies are described to evaluate the extent to which aggrephores recycle during continuous vasopressin stimulation or in a subsequent response following removal of hormone.
| Wade, James B; Liu, Jie; Coleman, Richard et al. (2015) SPAK-mediated NCC regulation in response to low-K+ diet. Am J Physiol Renal Physiol 308:F923-31 |
| Grimm, P Richard; Lazo-Fernandez, Yoskaly; Delpire, Eric et al. (2015) Integrated compensatory network is activated in the absence of NCC phosphorylation. J Clin Invest 125:2136-50 |
| Li, Lijun; Garikepati, R Mayuri; Tsukerman, Susanna et al. (2013) Reduced ENaC activity and blood pressure in mice with genetic knockout of the insulin receptor in the renal collecting duct. Am J Physiol Renal Physiol 304:F279-88 |
| Grimm, P Richard; Taneja, Tarvinder K; Liu, Jie et al. (2012) SPAK isoforms and OSR1 regulate sodium-chloride co-transporters in a nephron-specific manner. J Biol Chem 287:37673-90 |
| Wade, James B; Fang, Liang; Coleman, Richard A et al. (2011) Differential regulation of ROMK (Kir1.1) in distal nephron segments by dietary potassium. Am J Physiol Renal Physiol 300:F1385-93 |
| Liu, Wen; Schreck, Carlos; Coleman, Richard A et al. (2011) Role of NKCC in BK channel-mediated net K? secretion in the CCD. Am J Physiol Renal Physiol 301:F1088-97 |
| Wade, James B (2011) Statins affect AQP2 traffic. Am J Physiol Renal Physiol 301:F308 |
| Welling, Paul A; Chang, Yen-Pei C; Delpire, Eric et al. (2010) Multigene kinase network, kidney transport, and salt in essential hypertension. Kidney Int 77:1063-9 |
| Wang, Ying; O'Connell, Jeffrey R; McArdle, Patrick F et al. (2009) From the Cover: Whole-genome association study identifies STK39 as a hypertension susceptibility gene. Proc Natl Acad Sci U S A 106:226-31 |
| Fang, Liang; Garuti, Rita; Kim, Bo-Young et al. (2009) The ARH adaptor protein regulates endocytosis of the ROMK potassium secretory channel in mouse kidney. J Clin Invest 119:3278-89 |
Showing the most recent 10 out of 56 publications
