The amiloride-sensitive sodium channel that is present in many epithelial organs plays a major role in maintaining electrolyte homeostsis in mammals. It appears responsible for fine regulation of sodium loss by kidney and intestine as it is uniquely capable of lowering luminal sodium concentrations to a few millimolar. Although considerable physiological and biophysical information is available, biochemical and cell biological information, e.g., about molecular weight, peptide composition, or biosynthesis, is sparse. This project focuses on the characterization of the biochemical properties of the channel in colonic brush border membranes. In the rat, the levels of this channel are under complete control of adrenal steroid hormones; no channels are detectable in adrenalectomized animals; high levels can be induced by administration of exogenous steroids, particularly in the distal colon. Therefore, colonic brush border membranes will be prepared from uninduced and induced animals and compared with respect to transport properties, biochemical composition, and antigenic determinants with particular emphasis on charcterization of the steroid-dependent components by 3 different methodologies: 1) Transport of sodium and chloride will be characterized in the isolated vesicle preparation with respect to mechanism and sensitivity to putative inhibitors. 2) Peptide composition will be analyzed by SDS-PAGE and Western blots. 3) Polyclonal antibodies will be prepared against the brush border membrane with an enrichment of antibodies against the steroid-induced antigens. This investigation should yield information on transport properties of the colonic brush border membranes in the basal and steroid-dependent state and the peptides involved in the amiloride-sensitive sodium channels, as well as provide a specific probe in terms of a polyclonal antiserum. The development of antibodies is considered essential as they provide the means for future cell physiological studies, preparative procedures, and analysis of levels of these sodium channels in human disease states.
| Subramanian, Savitha; Han, Chang Yeop; Chiba, Tsuyoshi et al. (2008) Dietary cholesterol worsens adipose tissue macrophage accumulation and atherosclerosis in obese LDL receptor-deficient mice. Arterioscler Thromb Vasc Biol 28:685-91 |
