These results suggest that not only the intestinal epithelium but also other tissues are responsive to dietary copper to regulate the abundance of the adult glycosylated form of the Ctr1 copper importer. == Determine 6. levels of both total and apical membrane Ctr1 protein in response to transient dietary copper limitation. Experiments in cultured HEK293T cells demonstrated that alterations in the levels of the glycosylated form of Ctr1 in response to copper availability were a time-dependent, copper-specific posttranslational response. Taken together, these results demonstrate O6-Benzylguanine apical localization of Ctr1 in intestinal epithelia across three mammalian species and suggest that increased Ctr1 apical localization in response to dietary copper limitation may symbolize an adaptive response to homeostatically modulate Ctr1 availability at the site of intestinal copper absorption. Keywords:Copper, Intestine, Mammal, Metals, Transport Metals == Introduction == Copper is an essential trace element that supports catalysis by a wide range of enzymatic activities that include lysyl oxidase, copper/zinc-superoxide dismutase, dopamine -monooxygenase, as well as others O6-Benzylguanine that carry Rabbit Polyclonal to ITCH (phospho-Tyr420) out key physiological functions (15). Mammalian copper deficiency leads to defects in growth, development, and cognition, and recent reports indicate that many patients undergoing bariatric surgery or with other conditions that lead to a copper deficiency may experience severe myeloneuropathy (6,7). Consequently, it is important to identify those components that carry out copper uptake, distribution, and utilization and to understand how these components are regulated in their large quantity, activity, or subcellular localization. Many proteins have been recognized that play important functions in mammalian copper homeostasis, yet we know relatively little about their detailed mechanisms of action in cultured cells or in unique tissues or cell typesin vivo(3,4,8). Previous studies show that dietary copper absorption occurs largely in the small intestine (9). Moreover, mammals preconditioned on a copper-deficient diet demonstrate strongly enhanced uptake of copper in a subsequent exposure compared with control subjects, suggesting a regulatory mechanism for controlling dietary copper uptake in response to copper status (1012). Even though mechanisms underlying this regulation are not comprehended, Ctr1 is a high-affinity copper(I) transporter that is conserved in overall structure and function from yeast to humans (3,13,14). In both yeast and cultured mammalian cells, Ctr1 has been shown to traffic from your plasma membrane to intracellular compartments both constitutively and in response to elevated copper levels, prompting speculation that intestinal Ctr1 may play a significant role in intestinal copper absorption and its regulation (1517). Mice with an intestinal epithelial cell (IEC)3-specific Ctr1 knock-out (Ctr1int/int) show severe copper deficiency in peripheral tissues and pass away 3 weeks after birth (18). The ability to rescueCtr1int/intmice with a single intraperitoneal copper injection to bypass intestinal absorption strongly suggests that intestinal epithelial Ctr1 plays a critical role in dietary copper absorption (18). Previous reports suggest that Ctr1 resides around the apical membrane and in intracellular vesicular compartments of rodent IEC (Refs.11,18, and19and reviewed in Ref.8). However, a recent statement by Zimnickaet al.(20) suggests that Ctr1 resides exclusively around the basolateral membrane of Caco-2 cells, a cell culture model for polarized epithelial cells, O6-Benzylguanine and on the basolateral membrane of mouse IEC. As copper acquisition is critical for normal growth, development, cognition, and neurological function (15) and Ctr1 plays a vital role in O6-Benzylguanine dietary copper absorption through the intestine (18), it is important to clearly establish the location of Ctr1 in IECin vivo. In this study, we demonstrate apical localization of Ctr1 in mouse, rat, and pig intestine using immunohistochemistry analysis. Moreover, intestinal luminal cell surface biotinylation and cell culture experiments demonstrate elevated levels of Ctr1 at the apical surface in response to a copper limitation and suggest that changes in the large quantity of apical Ctr1, at a posttranslational level, may contribute to the regulation of dietary copper uptake in the intestine as a function of copper status. == MATERIALS AND METHODS == == == == == == Antibodies == Generation of the anti-Ctr1 antibody was explained and characterized previously (18). The anti-Ctr1 amino-terminal antibody was a gift from Dr. Jack H. Kaplan, University of Illinois (Chicago, IL). The anti-hephaestin antibody (HEPH11-A) was purchased from Alpha Diagnostics (San Antonio, TX). The anti-sodium/potassium/chloride co-transporter antibody (NKCC; T4) was obtained from the Developmental Studies Hybridoma Bank at the Department of Biology, University of Iowa (Iowa City, IA). The anti-superoxide dismutase antibody (SOD1; SOD-100) was purchased from Assay Designs (Ann Arbor, MI). The anti-copper chaperone for superoxide dismutase antibody (CCS; FL274) was purchased from Santa.