May 16, 2007; doi:10.1152/ajprenal.00064.2007.-The distal convoluted tubule (DCT) Na ϩ -Cl Ϫ cotransporter (NCC), the target of thiazide diuretics, is responsible for the reabsorption of 5-10% of filtered NaCl. The aim of this study was to test the hypothesis that acute infusion of the angiotensin-converting enzyme (ACE) inhibitor captopril (at 12 g/min) for 20 min provokes trafficking of NCC from apical plasma membranes (APM) to subapical cytoplasmic vesicles (SCV), which is reversed by acute ANG II infusion (ANG II at 20 ng ⅐ kg Ϫ1 ⅐ min Ϫ1 along with 12 g/min captopril) for 20 min in male Sprague-Dawley rats (250 -350 g). By immuno-electron microscopy using an anti-NCC (D. Ellison) 71.5 Ϯ SD 4.9% of the NCC gold labeling was associated with the APM in control, sham operated, and infused rats, while captopril infusion reduced NCC in APM to 54.9 Ϯ 6.9% (P Ͻ 0.001) and markedly increased immunogold labeling of SCV. Subsequent infusion of ANG II with captopril restored NCC immunogold labeling of APM to 72.4 Ϯ 4.2%, that is, 20% of the total NCC trafficked between APM and SCV. Likewise, on density gradients of cortex, captopril provoked redistribution of 27.3% of total NCC from low-density APM-enriched membranes to higher-density membranes and ANG IIϩcaptopril restored 20.3% of the NCC to APM-enriched fractions. Redistribution occurred independent of a change in NCC total abundance. In conclusion, this study demonstrates that ACE inhibition provokes acute trafficking of NCC out of the plasma membrane, which likely decreases DCT Na ϩ reabsorption, while ANG II provokes rapid trafficking of NCC from stores in subapical vesicles to the plasma membrane, which likely increases DCT Na ϩ reabsorption. sodium transport; thiazide receptor; immunoelectron microscopy THE NA ϩ -CL Ϫ COTRANSPORTER (NCC) is expressed in the apical membrane of the distal convoluted tubule (DCT) and is the target of the thiazide diuretics, which are frequently used in the treatment of hypertension and edema (1, 29). The importance of NCC in the regulation of blood pressure and salt balance is demonstrated in the genetic disorder Gitelman's syndrome in which loss of function mutations in NCC results in salt wasting, hypokalemia, and hypotension (32). Studies in mice with NCC knocked out show that on low-sodium diets, blood pressure is significantly reduced from control (31).There are multiple mechanisms by which NCC could be regulated to control Na ϩ transport in the DCT. Many studies have shown that NCC abundance is regulated by stimuli, such as dietary salt, aldosterone escape, and mineralocorticoid receptor blockade (22,26,33,36), less is known about trafficking of NCC to and from the apical membrane as a way of regulating NCC. The potential importance of trafficking in the regulation of NCC has been demonstrated in vitro in Gitelman's syndrome where oocyte studies indicate that NCC is not processed properly in the endoplasmic reticulum, resulting in deficient trafficking of NCC to the plasma membrane (8,14). A previous study from this labora...

FXYD5 (related to ion channel, dysadherin) is a member of the FXYD family of single span type I membrane proteins. Five members of this group have been shown to interact with the Na,KATPase and to modulate its properties. However, FXYD5 is structurally different from other family members and has been suggested to play a role in regulating E-cadherin and promoting metastasis (Ino, Y., Gotoh, M., Sakamoto, M., Tsukagoshi, K., and Hirohashi, S. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 365-370). The goal of this study was to determine whether FXYD5 can modulate the Na,KATPase activity, establish its cellular and tissue distribution, and characterize its biochemical properties. Anti-FXYD5 antibodies detected a 24-kDa polypeptide that was preferentially expressed in kidney, intestine, spleen, and lung. In kidney, FXYD5 resides in the basolateral membrane of the connecting tubule, the collecting tubule, and the intercalated cells of the collecting duct. However, there is also labeling of the apical membrane in long thin limb of Henle's loop. FXYD5 was effectively immunoprecipitated by antibodies to the ␣ subunit of Na,K-ATPase and the anti-FXYD5 antibody immunoprecipitates ␣. Co-expressing FXYD5 with the ␣1 and ␤1 subunits of the Na,K-ATPase in Xenopus oocytes elicited a more than 2-fold increase in pump activity, measured either as ouabainblockable outward current or as ouabain-sensitive 86 Rb ؉ uptake.Thus, as found with other FXYD proteins, FXYD5 interacts with the Na,K-ATPase and modulates its properties.Work in several laboratories led to the identification of a family of proteins, named after the common motif FXYD (1). Five members of this group have been shown to interact with the Na,K-ATPase and elicit different effects on its kinetics. These are as follows: FXYD1 (phospholemman, PLM), 3 (2); FXYD2 (the ␥ subunit of Na,K-ATPase, ␥) (3); FXYD3 (Mat-8) (4); FXYD4 (corticosteroid hormone-induced factor, CHIF) (5); and FXYD7 (6). In addition, a PLM-like protein from shark rectal gland has been characterized (7,8). The remaining two family members FXYD5 (related to ion channel, dysadherin) and FXYD6 have not yet been analyzed for possible interactions with the Na,K-ATPase. The working hypothesis is that all family members modulate the pump kinetics in vivo and function as tissue-specific modulators of the Na,KATPase (9 -11). However, other functions for FXYD proteins have also been suggested (12-16).FXYD proteins are type I membrane proteins with an extracellular N terminus (sometimes including a signal peptide), a single transmembrane domain, and an intracellular C terminus. With the exception of FXYD5, the extracellular domain is shorter than 40 amino acids, including a cleavable signal peptide. In the case of FXYD5, the extracellular domain is long, Ͼ140 amino acids. On the other hand, FXYD5 has the shortest intracellular C-terminal segment of only 15 amino acids. FXYD5 has been cloned as a tissue-specific and developmentally regulated gene induced by the oncoprotein E2a-Pbx1 and termed "related to ion channel...

Yang LE, Sandberg MB, Can AD, Pihakaski-Maunsbach K, McDonough AA. Effects of dietary salt on renal Na ϩ transporter subcellular distribution, abundance, and phosphorylation status. Am J Physiol Renal Physiol 295: F1003-F1016, 2008. First published July 23, 2008 doi:10.1152/ajprenal.90235.2008 diet the kidney increases urinary Na ϩ and volume excretion to match intake. We recently reported that HS provokes a redistribution of distal convoluted tubule Na ϩ -Cl Ϫ cotransporter (NCC) from apical to subapical vesicles and decreases NCC abundance. This study aimed to test the hypothesis that the other renal Na ϩ transporters' abundance and or subcellular distribution is decreased by HS diet. Six-week-old Sprague-Dawley rats were fed a normal (NS) 0.4% NaCl diet or a HS 4% NaCl diet for 3 wk or overnight. Kidneys excised from anesthetized rats were fractionated on density gradients or analyzed by microscopy; transporters and associated regulators were detected with specific antibodies. Three-week HS doubled Na ϩ /H ϩ exchanger (NHE)3 phosphorylation at serine 552 and provoked a redistribution of NHE3, dipeptidyl peptidase IV (DPPIV), myosin VI, Na ϩ -Pi cotransporter (NaPi)-2, ANG II type 2 receptor (AT2R), aminopeptidase N (APN), Na ϩ -K ϩ -2Cl Ϫ cotransporter (NKCC2), epithelial Na ϩ channel (ENaC) ␤-subunit, and Na ϩ -K ϩ -ATPase (NKA) ␣1-and ␤ 1-subunits from low-density plasma membrane-enriched fractions to higher-density intracellular membrane-enriched fractions. NHE3, myosin VI, and AT 2R retraction to the base of the microvilli (MV) during HS was evident by confocal microscopy. HS did not change abundance of NHE3, NKCC, or NKA ␣ 1-or ␤1-subunits but increased ENaC-␤ in high-density intracellular enriched membranes. Responses to HS were fully apparent after just 18 h. We propose that retraction of NHE3 to the base of the MV, driven by myosin VI and NHE3 phosphorylation and accompanied by redistribution of the NHE3 regulator DPPIV, contributes to a decrease in proximal tubule Na ϩ reabsorption during HS and that redistribution of transporters out of low-density plasma membrane-enriched fractions in the thick ascending limb of the loop of Henle and distal nephron may also contribute to the homeostatic natriuretic response to HS diet. sodium chloride; kidney; natriuresis; salt-sensitive hypertension A HIGH DIETARY SODIUM LOAD provokes a thirst that increases extracellular fluid volume (ECFV), maintaining plasma Na ϩ concentration in a normal range (40). The kidneys affect natriuretic and diuretic responses to match salt and water output to intake and restore ECFV. If volume is not corrected, it provokes a generalized vascular contraction to normalize tissue blood flow, resulting in hypertension. Establishing the molecular mechanisms by which the kidney regulates Na ϩ and ECFV homeostasis under normal conditions is an important goal because it will identify candidates that may contribute to salt-sensitive hypertension (6). Na ϩ transport along the nephron can be regulated by altering 1) total transporter abundanc...

(ANG II) stimulates proximal tubule (PT) sodium and water reabsorption. We showed that treating rats acutely with the angiotensin-converting enzyme inhibitor captopril decreases PT salt and water reabsorption and provokes rapid redistribution of the Na ϩ /H ϩ exchanger isoform 3 (NHE3), Na ϩ /Pi cotransporter 2 (NaPi2), and associated proteins out of the microvilli. The aim of the present study was to determine whether acute ANG II infusion increases the abundance of PT NHE3, NaPi2, and associated proteins in the microvilli available for reabsorbing NaCl. Male Sprague-Dawley rats were infused with a dose of captopril (12 g/min for 20 min) that increased PT flow rate ϳ20% with no change in blood pressure (BP) or glomerular filtration rate (GFR). When ANG II (20 ng⅐kg Ϫ1 ⅐min Ϫ1 for 20 min) was added to the captopril infusate, PT volume flow rate returned to baseline without changing BP or GFR. After captopril, NHE3 was localized to the base of the microvilli and NaPi2 to subapical cytoplasmic vesicles; after 20 min ANG II, both NHE3 and NaPi2 redistributed into the microvilli, assayed by confocal microscopy and density gradient fractionation. Additional PT proteins that redistributed into low-density microvilli-enriched membranes in response to ANG II included myosin VI, DPPIV, NHERF-1, ezrin, megalin, vacuolar H ϩ -ATPase, aminopeptidase N, and clathrin. In summary, in response to 20 min ANG II in the absence of a change in BP or GFR, multiple proteins traffic into the PT brush-border microvilli where they likely contribute to the rapid increase in PT salt and water reabsorption. hypertension; captopril ANGIOTENSIN II (ANG II), a potent vasoconstrictor and sodiumretaining hormone, is crucial for the regulation of sodium transport in the kidneys and therefore for blood pressure (BP) homeostasis. Strong evidence highlights the contribution of high ANG II levels to the development of cardiovascular and renal diseases (23, 37). Consequently, drugs affecting the renin-angiotensin system (RAS), and in particular angiotensinconverting enzyme inhibitors (ACEI) and angiotensin receptor blockers, are commonly used for the treatment of high BP.The angiotensin type I receptor (AT1R) is responsible for the Na ϩ -retaining effects of ANG II. The renal proximal tubule (PT) expresses AT1R on both the apical and basolateral membranes and ANG II is delivered via the general circulation or filtered at the glomerulus. In addition, the PT cells synthesize all the components necessary to produce and secrete ANG II into its lumen: angiotensinogen, renin, and ACE (14, 29). ANG II has been shown to increase PT sodium and water reabsorption, and ACE inhibitors and AT1R blockers decrease PT sodium and water reabsorption (10, 13). The sodium hydrogen exchanger isoform 3 (NHE3) is the main transporter mediating sodium reabsorption in the PT (20) and cultured kidney cell studies indicate that ANG II can rapidly increase NHE3 abundance and activity in the plasma membrane (9).We recently investigated the molecular mechanisms responsible for th...

No abstract

Six antifreeze proteins, which have the unique ability to adsorb onto the surface of ice and inhibit its growth, have been isolated from the apoplast of winter rye leaves where ice forms at subzero temperatures. The rye antifreeze proteins accumulate during cold acclimation and are similar to plant pathogenesis‐related proteins, including two endoglucanase‐like, two chitinase‐like and two thaumatin‐like proteins. Immunolocalization of the glucanase‐like antifreeze proteins showed that they accumulate in mesophyll cell walls facing intercellular spaces, in pectinaceous regions between adjoining mestome sheath cells, in the secondary cell walls of xylem vessels and in epidermal cell walls. Because the rye antifreeze proteins are located in areas where they could be in contact with ice, they may function as a barrier to the propagation of ice or to inhibit the recrystallization of ice. Antifreeze proteins similar to pathogenesis‐related proteins were also found to accumulate in closely‐related plants within the Triticum group but not in freezing‐tolerant dicotyledonous plants. In winter wheat, the accumulation of antifreeze proteins and the development of freezing tolerance are regulated by chromosome 5. Rye antifreeze proteins may have evolved from pathogenesis‐related proteins, but they retain their catalytic activities and may play a dual role in increasing both freezing and disease resistance in overwintering plants.

Nodulin genes are specifically expressed in the nitrogen-fixing root nodules. We have identified a novel type of DNA-binding protein (CPP1) interacting with the promoter of the soybean leghemoglobin gene Gmlbc3. The DNA-binding domain of CPP1 contains two similar Cys-rich domains with 9 and 10 Cys, respectively. Genes encoding similar domains have been identified in Arabidopsis thaliana, Caenorhabditis elegans, the mouse, and human. The domains also have some homology to a Cys-rich region present in some polycomb proteins. The cpp1 gene is induced late in nodule development and the expression is confined to the distal part of the central infected tissue of the nodule. A constitutively expressed cpp1 gene reduces the expression of a Gmlbc3 promoter-gusA reporter construct in Vicia hirsuta roots. These data therefore suggest that CPP1 might be involved in the regulation of the leghemoglobin genes in the symbiotic root nodule.

During cold acclimation, antifreeze proteins (AFPs) that are similar to pathogenesis-related proteins accumulate in the apoplast of winter rye (Secale cereale L. cv Musketeer) leaves. AFPs have the ability to modify the growth of ice. To elucidate the role of AFPs in the freezing process, they were assayed and immunolocalized in winter rye leaves, crowns, and roots. Each of the total soluble protein extracts from cold-acclimated rye leaves, crowns, and roots exhibited antifreeze activity, whereas no antifreeze activity was observed in extracts from nonacclimated rye plants. Antibodies raised against three apoplastic rye AFPs, corresponding to a glucanase-like protein (CLP, 32 kD), a chitinase-like protein (CLP, 35 kD), and a thaumatin-like protein (TLP, 25 kD), were used in tissue printing to show that the AFPs are localized in the epidermis and in cells surrounding intercellular spaces in cold-acclimated plants. Although GLPs, CLPs, and TLPs were present in nonacclimated plants, they were found in different locations and did not exhibit antifreeze activity, which suggests that different isoforms of pathogenesis-related proteins are' produced at low temperature. The location of rye AFPs may prevent secondary nucleation of cells by epiphytic ice or by ice propagating through the xylem. l h e distributions of pathogenesis-induced and cold-accumulated CLPs, CLPs, and TLPs are similar and may reflect the common pathways by which both pathogens and ice enter and propagate through plant tissues.