Levodopa (L-DOPA) is the naturally occurring precursor amino acid for dopamine and the main therapeutic agent for neurologic disorders due to dopamine depletion, such as Parkinson's disease. L-DOPA absorption in small intestine has been suggested to be mediated by the large neutral amino acids transport machinery, but the identity of the involved transporters is unknown. Clinically, coadministration of L-DOPA and dietary amino acids is avoided to decrease competition for transport in intestine and at the blood-brain barrier. L-DOPA is routinely coadministered with levodopa metabolism inhibitors (dopa-decarboxylase and cathechol-O-methyl transferase inhibitors) that share structural similarity with levodopa. In this systematic study involving Xenopus laevis oocytes and Madin-Darby canine kidney epithelia expression systems and ex vivo preparations from wild-type and knockout mice, we identified the neutral and dibasic amino acids exchanger (antiporter) b(0,+)AT-rBAT (SLC7A9-SLC3A1) as the luminal intestinal L-DOPA transporter. The major luminal cotransporter (symporter) B(0)AT1 (SLC6A19) was not involved in levodopa transport. L-Leucine and L-arginine competed with levodopa across the luminal enterocyte membrane as expected for b(0,+)AT-rBAT substrates, whereas dopadecarboxylase and cathechol-O-methyl transferase inhibitors had no effect. The presence of amino acids in the basolateral compartment mimicking the postprandial phase increased transepithelial levodopa transport by stimulating basolateral efflux via the antiporter LAT2-4F2 (SLC7A8-SLC3A2). Additionally, the aromatic amino acid uniporter TAT1 (SLC16A10) was shown to play a major role in L-DOPA efflux from intestinal enterocytes. These results identify the molecular mechanisms mediating small intestinal levodopa absorption and suggest strategies for optimization of delivery and absorption of this important prodrug.

Une neurotoxine, inhibant la synthèse protéique dans des cellules en culture, a été isolée d'écorces de racines de Cnestis glabra (Connaracées) avec un rendement de l'ordre de 0.4 pour cent. Le procédé de purification utilisé comporte 5 étapes : filtration sur une couche de charbon actif, traitement par l'acétate neutre de plomb et fractionnements par chromatographies successives sur Dowex 50 × 8 sous forme H+ et NH4+. L'homogénéité de la toxine purifiée a été vérifiée par chromatographies sur couche mince et en phase gazeuse. Son poids moléculaire est inférieur à 500. C'est un composé thermostable, insoluble dans les solvants organiques usuels et qui donne une réaction positive à la ninhydrine. L'hydrolyse acide ne modifie pas son comportement sur un analyseur d'acides aminés. Sa nature possible (acide aminé) est discutée. La toxine est provisoirement appeléc glabrine.A neurotoxic compound, inhibiting protein synthesis in cell culture, was isolated in a yield of about 0.4 per cent from Cnestis glabra root barks (Connaraceae) by a five-step fractionation procedure (filtration on activated charcoal, treatment by neutral lead acetate and fractionations on Dowex 50 × 8 in H+ and NH4+ forms). The purified toxin appeared homogeneous on thin-layer and in gas chromatography. The compound has a low molecular wweight (less than 500). It is heat-stable, insoluble in usual organic solvents and gives a positive reaction with ninhydrin. Acidic hydrolysis does not change its behaviour on an amino acid analyzer. Its possible amino acid nature is discussed. It is temporarily named glabrin.

Purified mouse plasmocytoma ribosomes uncontaminated with free mRNP particles were prepared. After treatment with 0.5 M KCl a complex containing hoth dRNA and proteins was released (FKCl). This contained nearly 6 p. 100 dRNA; in the protein fraction the presence of phosphoproteins was detected. The density of this structure was 1.18 g/ml in a sucrose gradient and 1.3 g/ml in a CsCl gradient. This material was different from the material released from ribosomes after EDTA treatment (polysomal mRNP). The electrophoretic protein patterns in SDS-polyacrylamide gels showed three main protein bands of high molecular weight (89,000, 100,000 and 105,000 dalton) and also some minor bands. The addition of the «FKCl fraction to a cell-free system containing washed KCl ribosomes, supernatant fraction S200 and polyU, markedly stimulated the incorporation of 14C phenylalanine into polypeptides.The possibility of an association between one or another initiation factors with the initiation sites present on mRNA s suggested.Des ribosomes purifiés de plasmocytome de souris non contaminés par des particules mRNP libres ont été préparés. Après un traitement par du KCl 0,5 M on a détaché un complexe contenant à la fois du dRNA et des protéines («F KCl). Il contient environ 6 p. cent de dRNA et parmi les protéines on a mis en évidence la présence de phosphoprotéines. Cette structure sédimente avec une densité à l'équilibre de 1,18 en gradient de saccharose et de 1,3 en CsCl. Ce matériel est différent de celui détaché des ribosomes sous l'effet de l'EDTA (mRNP polysomiques). L'analyse du profil électrophorétique des protéines sur gel de polyacrylamide-SDS révèle trois bandes protéiques majeures de haut poids moléculaire (89.000, 100.000, 105.000) et plusieurs bandes mineures. Additionné à un système acellulaire contenant la fraction soluble S200, des ribosomes lavés au KCl et du polyU, le complexe «F KCl provoque une stimulation importante de l'incorporation de 14C-phénylalanine dans les polypeptides.La possibilité d'une association de l'un ou l'autre des facteurs d'initiation avec les sites d'initiation portés par le mRNA est suggérée.

Mutations in the main intestinal and kidney luminal neutral amino acid transporter B(0)AT1 (Slc6a19) lead to Hartnup disorder, a condition that is characterized by neutral aminoaciduria and in some cases pellagra-like symptoms. These latter symptoms caused by low-niacin are thought to result from defective intestinal absorption of its precursor L-tryptophan. Since Ace2 is necessary for intestinal B(0)AT1 expression, we tested the impact of intestinal B(0)AT1 absence in ace2 null mice. Their weight gain following weaning was decreased, and Na(+)-dependent uptake of B(0)AT1 substrates measured in everted intestinal rings was defective. Additionally, high-affinity Na(+)-dependent transport of L-proline, presumably via SIT1 (Slc6a20), was absent, whereas glucose uptake via SGLT1 (Slc5a1) was not affected. Measurements of small intestine luminal amino acid content following gavage showed that more L-tryptophan than other B(0)AT1 substrates reach the ileum in wild-type mice, which is in line with its known lower apparent affinity. In ace2 null mice, the absorption defect was confirmed by a severalfold increase of L-tryptophan and of other neutral amino acids reaching the ileum lumen. Furthermore, plasma and muscle levels of glycine and L-tryptophan were significantly decreased in ace2 null mice, with other neutral amino acids displaying a similar trend. A low-protein/low-niacin diet challenge led to differential changes in plasma amino acid levels in both wild-type and ace2 null mice, but only in ace2 null mice to a stop in weight gain. Despite the combination of low-niacin with a low-protein diet, plasma niacin concentrations remained normal in ace2 null mice and no pellagra symptoms, such as photosensitive skin rash or ataxia, were observed. In summary, mice lacking Ace2-dependent intestinal amino acid transport display no total niacin deficiency nor clear pellagra symptoms, even under a low-protein and low-niacin diet, despite gross amino acid homeostasis alterations.

Mutations in angiotensin-converting enzyme 2 are shown to predispose mice to colitis as a consequence of neutral amino acid malabsorption and a change in the resident microbiota; these results could explain how protein malnutrition — affecting up to one billion people — leads to intestinal inflammation. Malnutrition affects many millions of people in the developing world and remains a problem in wealthy nations, especially for disadvantaged groups. In many cases, it is the associated diarrhoea and intestinal inflammation that cause morbidity and death. A study published in this issue presents a molecular explanation for the increased susceptibility to intestinal inflammation in malnutrition. Angiotensin converting enzyme 2 (ACE2), which has a central role in blood-pressure regulation and has been implicated in diabetes, heart failure and viral infection, is shown to influence dietary amino-acid homeostasis, innate immunity, gut microbial ecology and susceptibility to colitis. Mice deficient in this enzyme show impaired tryptophan metabolism and develop colitis, which is alleviated by dietary tryptophan and its metabolite, nicotinamide. This surprising result explains nutritional effects that have been known for centuries and provides a molecular link between malnutrition and the intestinal microbiome. Malnutrition affects up to one billion people in the world and is a major cause of mortality1,2. In many cases, malnutrition is associated with diarrhoea and intestinal inflammation, further contributing to morbidity and death2. The mechanisms by which unbalanced dietary nutrients affect intestinal homeostasis are largely unknown. Here we report that deficiency in murine angiotensin I converting enzyme (peptidyl-dipeptidase A) 2 (Ace2), which encodes a key regulatory enzyme of the renin-angiotensin system (RAS), results in highly increased susceptibility to intestinal inflammation induced by epithelial damage. The RAS is known to be involved in acute lung failure3, cardiovascular functions4 and SARS infections5. Mechanistically, ACE2 has a RAS-independent function, regulating intestinal amino acid homeostasis, expression of antimicrobial peptides, and the ecology of the gut microbiome. Transplantation of the altered microbiota from Ace2 mutant mice into germ-free wild-type hosts was able to transmit the increased propensity to develop severe colitis. ACE2-dependent changes in epithelial immunity and the gut microbiota can be directly regulated by the dietary amino acid tryptophan. Our results identify ACE2 as a key regulator of dietary amino acid homeostasis, innate immunity, gut microbial ecology, and transmissible susceptibility to colitis. These results provide a molecular explanation for how amino acid malnutrition can cause intestinal inflammation and diarrhoea.

The uniporter TAT1 (Slc16a10) mediates the facilitated diffusion of aromatic amino acids (AAAs) across basolateral membranes of kidney, small intestine and liver epithelial cells, and across the plasma membrane of non-epithelial cells like skeletal myocytes. Its role for body AA homeostasis has now been investigated using newly generated TAT1 (Slc16a10) defective mice (tat1(-/-)). These mice grow and reproduce normally, show no gross phenotype and no obvious neurological defect. Histological analysis did not reveal abnormalities and there is no compensatory change in any tested AA transporter mRNA. TAT1 null mice, however, display increased plasma, muscle and kidney AAA concentration under both normal and high protein diet, although this concentration remains normal in the liver. A major aromatic aminoaciduria and a smaller urinary loss of all substrates additionally transported by l-type AA antiporter Lat2-4F2hc (Slc7a8) were revealed under a high protein diet. This suggests an epithelial transport defect as also shown by the accumulation of intravenously injected (123)I-2-I-l-Phe in kidney and l-[(3)H]Phe in ex vivo everted gut sac enterocytes. Taken together, these data indicate that the uniporter TAT1 is required to equilibrate the concentration of AAAs across specific membranes. For instance, it enables hepatocytes to function as a sink that controls the extracellular AAAs concentration. Additionally, it facilitates the release of AAAs across the basolateral membrane of small intestine and proximal kidney tubule epithelial cells, thereby allowing the efflux of other neutral AAs presumably via Lat2-4F2hc.

We have previously demonstrated that high amino acids (AA) and insulin are required for stimulation of translation in hepatocytes. Three hepatic sensing pathways are regulated by dietary protein, i.e. mTOR, GCN2 and AMPK. However, the nature of the AA signal is still unknown. We aimed to identify this signal and clarify the relationship between the transduction pathways using primary hepatocyte culture. We examined the role of branched‐chain amino acids (BCAA) and L‐leucine (Leu) in these pathways. With similar findings to the ones we obtained upon high AA treatment, BCAA or Leu stimulated phosphorylation of mTOR, 4E‐BP1 and S6K1 and decreased phosphorylation of AMPK and GCN2. We further characterized the roles of mTOR and AMPK in the pathway by using AICAR, an activator of AMPK, and rapamycin, an inhibitor of mTOR. Our results show that AICAR not only induced AMPK phosphorylation with or without insulin, but also decreased mTOR phosphorylation. Rapamycin treatment further decreased mTOR phosphorylation. Surprisingly, 4E‐BP1, a well known target of mTOR was dramatically decreased by AICAR, but not by rapamycin. These results suggest that BCAA and Leu are the key amino acids sensed by hepatocytes, and that AMPK may act as a general switch during high AA thereby controlling protein synthesis. To characterize the hierarchy of the system more in detail we next aim to identify the protein phosphatases induced by high AA.

Neutral amino acid transporters of the SLC6 family are expressed at the apical membrane of kidney and/or small intestine, where they (re-)absorb amino acids into the body. In this review we present the results concerning the dependence of their apical expression with their association to partner proteins. We will in particular focus on the situation of B0AT1 and B0AT3, that associate with members of the renin-angiotensin system (RAS), namely Tmem27 and angiotensin-converting enzyme 2 (ACE2), in a tissue specific manner. The role of this association in relation to the formation of a functional unit related to Na+ or amino acid transport will be assessed. We will conclude with some remarks concerning the relevance of this association to Hartnup disorder, where some mutations have been shown to differentially interact with the partner proteins.

The basolateral amino acid transporters LAT2- and y+LAT1-4F2hc function as obligatory exchangers whereas the aromatic amino acid transporter TAT1 is known to function as a low affinity facilitated diffusion pathway. Using X. laevis oocytes we have shown that TAT1 can complement the transport function of LAT2-4F2hc by recycling exchange substrates and thus contribute to net amino acid (re)absorption. Under normal conditions, Tat1 knockout mice (Ingenium Pharmaceuticals) grow normally and show no gross phenotype: they exhibit increased plasma levels of Tyr and Trp, but no major aminoaciduria. However, when subjected to high protein diet, their urine contains several amino acids, whereas their plasma concentration remains stable. This data show that the lack of Tat1 impacts on the kinetics of net transepithelial (re)absorption of amino acids and their homeostasis. The lack of aminoaciduria observed under normal diet implies that at least one additional amino acid transporter capable of recycling exchange substrates is expressed in the same basolateral epithelial membranes. Nevertheless, as shown under high protein diet, this additional transporter(s) does not entirely compensate the absence of Tat1. Supported by Swiss NSF grant 31-130471 to François Verrey

Near complete reabsorption of filtered amino acids is a main specialized transport function of the kidney proximal tubule. This evolutionary conserved task is carried out by a subset of luminal and basolateral transporters that together form the transcellular amino acid transport machinery similar to that of small intestine. A number of other amino acid transporters expressed in the basolateral membrane of proximal kidney tubule cells subserve either specialized metabolic functions, such as the production of ammonium, or are part of the cellular housekeeping equipment. A new finding is that the luminal Na+-dependent neutral amino acid transporters of the SLC6 family require an associated protein for their surface expression as shown for the Hartnup transporter B0AT1 (SLC6A19) and suggested for the l-proline transporter SIT1 (IMINOB, SLC6A20) and for B0AT3 (XT2, SLC6A18). This accessory subunit called collectrin (TMEM27) is homologous to the transmembrane anchor region of the renin–angiotensin system enzyme ACE2 that we have shown to function in small intestine as associated subunit of the luminal SLC6 transporters B0AT1 and SIT1. Some mutations of B0AT1 differentially interact with these accessory subunits, providing an explanation for differential intestinal phenotypes among Hartnup patients. The basolateral efflux of numerous amino acids from kidney tubular cells is mediated by heteromeric amino acid transporters that function as obligatory exchangers. Thus, other transporters within the same membrane need to mediate the net efflux of exchange substrates, controlling thereby the net basolateral amino transport and thus the intracellular amino acid concentration.

* These authors contributed equally to this work 1 Institute of Physiology and Center for Integrative Human Physiology (ZIHP), University of Zürich, Switzerland 2 Institute for Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna, Austria 3 Functional Genomics Center of Zurich (FGCZ), University and ETH Zurich, Switzerland 4 Department of Cell Biology, Medicine and Neurobiology, Duke University Medical Center, Durham, USA

The orphan transporter Slc6a18 (XT2) is highly expressed at the luminal membrane of kidney proximal tubules and displays approximately 50% identity with Slc6a19 (B(0)AT1), which is the main neutral amino acid transporter in both kidney and small intestine. As yet, the amino acid transport function of XT2 has only been experimentally supported by the urinary glycine loss observed in xt2 null mice. We report here that in Xenopus laevis oocytes, co-expressed ACE2 (angiotensin-converting enzyme 2) associates with XT2 and reveals its function as a Na(+)- and Cl(-)-de pend ent neutral amino acid transporter. In contrast to its association with ACE2 observed in Xenopus laevis oocytes, our experiments with ace2 and collectrin null mice demonstrate that in vivo it is Collectrin, a smaller homologue of ACE2, that is required for functional expression of XT2 in kidney. To assess the function of XT2 in vivo, we reanalyzed its knock-out mouse model after more than 10 generations of backcrossing into C57BL/6 background. In addition to the previously published glycinuria, we observed a urinary loss of several other amino acids, in particular beta-branched and small neutral ones. Using telemetry, we confirmed the previously described link of XT2 absence with hypertension but only in physically restrained animals. Taken together, our data indicate that the formerly orphan transporter XT2 functions as a sodium and chloride-de pend ent neutral amino acid transporter that we propose to rename B(0)AT3.

Background & Aims: Hartnup amino acid transporter B(0)AT1 (SLC6A19) is the major luminal sodium-dependent neutral amino acid transporter of small intestine and kidney proximal tubule. The expression of B(0)AT1 in kidney was recently shown to depend on its association with collectrin (Tmem27), a protein homologous to the membrane-anchoring domain of angiotensin-converting enzyme (ACE) 2. Methods: Because collectrin is almost absent from small intestine, we tested the hypothesis that it is ACE2 that interacts with B(0)AT1 in enterocytes. Furthermore, because B(0)AT1 expression depends on an associated protein, we tested the hypothesis that Hartnup-causing B(0)AT1 mutations differentially impact on B(0)AT1 interaction with intestinal and kidney accessory proteins. Results: Immunofluorescence, coimmunoprecipitation, and functional experiments using wild-type and ace2-null mice showed that expression of B(0)AT1 in small intestine critically depends on ACE2. Coexpressing new and previously identified Hartnup, disorder-causing missense mutations of B(0)AT1 with either collectrin or ACE2 in Xenopus laevis oocytes showed that the high-frequency D173N and the newly identified P26SL mutant B(0)AT1 transporters can still be activated by ACE2 but not collectrin coexpression. In contrast, the human A69T and R240Q B(0)AT1 mutants cannot be activated by either of the associated proteins, although they function as wild-type B(0)AT1 when expressed alone. Conclusions: We thus show that ACE2 is necessary for the expression of the Hartnup transporter in intestine and suggest that the differential functional. association of mutant B(0)AT1 transporters with ACE2 and collectrin in intestine and kidney, respectively, participates in the phenotypic heterogeneity of human Hartnup disorder.

The antidiuretic effect of vasopressin is mediated by V2 receptors (V2R) that are located in kidney connecting tubules and collecting ducts. This study provides evidence that V2R signaling is negatively regulated by regulator of G protein signaling 2 (RGS2), a member of the family of RGS proteins. This study demonstrates that (1) RGS2 expression in the kidney is restricted to the vasopressin-sensitive part of the nephron (thick ascending limb, connecting tubule, and collecting duct); (2) expression of RGS2 is rapidly upregulated by vasopressin; (3) the vasopressin-dependent accumulation of cAMP, the principal messenger of V2R signaling, is significantly higher in collecting ducts that are microdissected from the RGS2(-/-) mice compared with their wild-type littermates; and (4) analysis of urine output of mice that were exposed to water restriction followed by acute water loading revealed that RGS2(-/-) mice exhibit an increased renal responsiveness to vasopressin. It is proposed that RGS2 is involved in negative feedback regulation of V2R signaling.

Angiotensin -converting enzyme 2 (ACE2) is a regulator of the renin angiotensin system involved in acute lung failure, cardiovascular functions and severe acute respiratory syndrome (SARS) infections in mammals1,2,3. A gene encoding a homologue to ACE2, termed collectrin (Tmem27), has been identified in immediate proximity to the ace2 locus4. The in vivo function of collectrin was unclear. Here we report that targeted disruption of collectrin in mice results in a severe defect in renal amino acid uptake owing to downregulation of apical amino acid transporters in the kidney. Collectrin associates with multiple apical transporters and defines a novel group of renal amino acid transporters. Expression of collectrin in Xenopus oocytes and Madin–Darby canine kidney (MDCK) cells enhances amino acid transport by the transporter B0AT1. These data identify collectrin as a key regulator of renal amino acid uptake.