Guanine nucleotide-dependent carboxymethylation: A pathway for aldosterone modulation of apical Na+ permeability in epithelia

A number of recent reviews have dealt extensively with the characteristics of epithelial Na+ channels and their classification according to level of conductance, selectivity for sodium, and sensitivity to amiloride and hormones [1 to 6, this issue]. In the present review, we will focus on Na+ transport regulation by aldosterone in terms of the biochemical pathways involved in the hormone's action on apical Na+ permeability in epithelia.Active transport of Na+ across epithelial tissues is the primary physiological process responsible for maintenance of salt balance in vertebrates. Entry of Na+ into cells at the apical membrane occurs passively by electrodiffusion through amiloride-blockable channels. Intracellular Na+ concentration remains low due to active extrusion across the basolateral membrane in exchange for K+. Aldosterone is the key hormone for long-term regulation of this process in the distal tubule of the kidney and other responsive “high resistance” model epithelia such as toad urinary bladder, frog skin, and A6 cultured cells derived from toad kidney [7, 8]. Its mode of action is complex involving a number of biochemical pathways, and some or possibly all of them ultimately increase the transepithelial Na+ transport rate. Aldosterone increases both the apical Na+ permeability and the number of basolateral pump sites albeit with different time courses and through distinct pathways [9–12].The increase of apical Na+ permeability in response to aldosterone is due to an increase in the number of open Na+ channels in this membrane with virtually no change in channel selectivity or conductance [10, 13]. Stimulation of transport begins after a lag time of 20 to 40 minutes and is maximal within four to six hours of exposure to the hormone. Aldosterone, a steroid hormone, enters cells by diffusion, is bound by an intracellular receptor, migrates to the nucleus, and gives rise to a variety of gene products commonly termed AIPs (aldosterone-induced proteins; cf. Fig. 1), which in turn lead to the various physiological responses. This scheme is supported by observations that increased Na+ transport after aldosterone is abolished by inhibitors of protein or RNA synthesis [14]. It is currently unknown whether any of the AIPs represent apical Na+ channels per se or subunits of these channels. This seems unlikely however in view of overwhelming electrical and biochemical data consistent with the idea that aldosterone stimulates channels pre-existing in the membrane [13, 15–18]. In addition, membrane targeting, channel assembly from subunits, and insertion of these complex molecules into the membrane, would likely take several hours as observed for the basolateral response where it has been demonstrated that pumps are synthesized and inserted over many hours or days [11, 12]. This would seem less plausible at the apical membrane where the more rapid initial increase in permeability occurs within 30 minutes. However, a polypeptide of Mr 70 kD, identified as a component of the Na+ channel, was shown to be induced by aldosterone [19]. Whether this polypeptide represents part of the channel or a regulatory protein is presently unknown.The sequence of events leading to increased channel density following synthesis of AIPs is not understood. However, a number of mechanisms modulate or mediate aldosterone's stimulation of apical Na+ permeability. (1) Activation of phospholipase A increases phospholipid fatty acid metabolism. (2) Methylation of apical proteins and lipids increases amiloride-sensitive Na+ transport. (3) Guanine nucleotides regulate the aldosterone-induced carboxymethylation.These pathways are not mutually exclusive and indeed may represent only a small portion of the complete picture of aldosterone's influence at the apical membrane. Each of these is considered separately below

Characterization of the Metabolic Phenotype of Rapamycin-Treated CD8+ T Cells with Augmented Ability to Generate Long-Lasting Memory Cells

Cellular metabolism plays a critical role in regulating T cell responses and the development of memory T cells with long-term protections. However, the metabolic phenotype of antigen-activated T cells that are responsible for the generation of long-lived memory cells has not been characterized.. than untreated control T cells. In contrast to that control T cells only increased glycolysis, rapamycin-treated T cells upregulated both glycolysis and oxidative phosphorylation (OXPHOS). These rapamycin-treated T cells had greater ability than control T cells to survive withdrawal of either glucose or growth factors. Inhibition of OXPHOS by oligomycin significantly reduced the ability of rapamycin-treated T cells to survive growth factor withdrawal. This effect of OXPHOS inhibition was accompanied with mitochondrial hyperpolarization and elevation of reactive oxygen species that are known to be toxic to cells.Our findings indicate that these rapamycin-treated T cells may represent a unique cell model for identifying nutrients and signals critical to regulating metabolism in both effector and memory T cells, and for the development of new methods to improve the efficacy of adoptive T cell cancer therapy

Epithelial sodium channel activity in detergent-resistant membrane microdomains.

The activity of epithelial Na(+) selective channels is modulated by various factors, with growing evidence that membrane lipids also participate in the regulation. In the present study, Triton X-100 extracts of whole cells and of apical membrane-enriched preparations from cultured A6 renal epithelial cells were floated on continuous-sucrose-density gradients. Na(+) channel protein, probed by immunostaining of Western blots, was detected in the high-density fractions of the gradients (between 18 and 30% sucrose), which contain the detergent-soluble material but also in the lighter, detergent-resistant 16% sucrose fraction. Single amiloride-sensitive Na(+) channel activity, recorded after incorporation of reconstituted proteoliposomes into lipid bilayers, was exclusively localized in the 16% sucrose fraction. In accordance with other studies, high- and low-density fractions of sucrose gradients likely represent membrane domains with different lipid contents. However, exposure of the cells to cholesterol-depleting or sphingomyelin-depleting agents did not affect transepithelial Na(+) current, single-Na(+) channel activity, or the expression of Na(+) channel protein. This is the first reconstitution study of native epithelial Na(+) channels, which suggests that functional channels are compartmentalized in discrete domains within the plane of the apical cell membrane.Journal ArticleResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe

Saturation behavior of single, amiloride-sensitive Na+ channels in planar lipid bilayers.

Single epithelial Na+ channels incorporated into planar lipid bilayers were studied to determine the effects of Na concentration on its own conductance. Amiloride-sensitive Na+ channels were obtained from apical membrane vesicles made from A6 cells, a continuous epithelial cell-line derived from amphibian kidney. Single-channel conductance was found to be a saturable function of Na+ concentration, with a Michaelis constant of approximately 17 or 47 mM, for a Gmax of approximately 4 or 44 pS, respectively