Decoding the substrate supply to human neuronal nitric oxide synthase

Nitric oxide, produced by the neuronal nitric oxide synthase (nNOS) from L-arginine is an important second messenger molecule in the central nervous system: It influences the synthesis and release of neurotransmitters and plays an important role in long-term potentiation, long-term depression and neuroendocrine secretion. However, under certain pathological conditions such as Alzheimer's or Parkinson's disease, stroke and multiple sclerosis, excessive NO production can lead to tissue damage. It is thus desirable to control NO production in these situations. So far, little is known about the substrate supply to human nNOS as a determinant of its activity. Measuring bioactive NO via cGMP formation in reporter cells, we demonstrate here that nNOS in both, human A673 neuroepithelioma and TGW-nu-I neuroblastoma cells can be fast and efficiently nourished by extracellular arginine that enters the cells via membrane transporters (pool I that is freely exchangeable with the extracellular space). When this pool was depleted, NO synthesis was partially sustained by intracellular arginine sources not freely exchangeable with the extracellular space (pool II). Protein breakdown made up by far the largest part of pool II in both cell types. In contrast, citrulline to arginine conversion maintained NO synthesis only in TGW-nu-I neuroblastoma, but not A673 neuroepithelioma cells. Histidine mimicked the effect of protease inhibitors causing an almost complete nNOS inhibition in cells incubated additionally in lysine that depletes the exchangeable arginine pool. Our results identify new ways to modulate nNOS activity by modifying its substrate supply

Gene expression during osteogenic differentiation in mandibular condyles in vitro

Abstract. The cartilagenous tissue of mandibular condyles of newborn mice contains progenitor cells as well as young and mature chondrogenic cells. During in vitro cultivation of the tissue, progenitor cells undergo osteogenic differentiation and form new bon

Voltage dependence of l

Nawrath H, Wegener J, Rupp J, Habermeier A, Closs EI. Voltage dependence of -arginine transport by hCAT-2A and hCAT-2B expressed in oocytes from. American Journal of Physiology-Cell Physiology. 2000;279(5):C1336-C1344.Membrane potential and currents were investigated with the two-electrode voltage-clamp technique in Xenopus laevisoocytes expressing hCAT-2A or hCAT-2B, the splice variants of the human cationic amino acid transporter hCAT-2. Both hCAT-2A- and hCAT-2B-expressing oocytes exhibited a negative extracellularl-arginine concentration ([l-Arg]o)-sensitive membrane potential, additive to the K+diffusion potential, when cells were incubated in Leibovitz medium (containing 1.45 mM l-Arg and 0.25 mM l-lysine). The two carrier proteins produced inward and outward currents, which were dependent on the l-Arg gradient and membrane potential. Ion substitution experiments showed that the hCAT-induced currents were independent of external Na+, K+, Ca2+, or Mg2+. The apparent Michaelis-Menten constant values at −60 mV, obtained from plots of l-Arg-induced currents against [l-Arg]o, were 0.97 and 0.13 mM in oocytes expressing hCAT-2A and hCAT-2B, respectively; maximal currents amounted to −194 ± 8 and −84 ± 2 nA, respectively. At saturating [l-Arg]o, the current-voltage relationships of hCAT-2A-expressing oocytes became steeper, yielding an additional conductance up to 2 μS/oocyte, whereas those of hCAT-2B-expressing oocytes were simply shifted to the right, resulting in voltage-independent difference currents. The distinct electrochemical properties of the two isoforms of hCAT-2 are assumed to contribute differentially to the membrane transport and the maintenance of cationic amino acids in various tissues

Asymmetric dimethylarginine is transported by the mitochondrial carrier SLC25A2

Asymmetric dimethyl l-arginine (ADMA) is generated within cells and in mitochondria when proteins with dimethylated arginine residues are degraded. The aim of this study was to identify the carrier protein(s) that transport ADMA across the inner mitochondrial membrane. It was found that the recombinant, purified mitochondrial solute carrier SLC25A2 when reconstituted into liposomes efficiently transports ADMA in addition to its known substrates arginine, lysine, and ornithine and in contrast to the other known mitochondrial amino acid transporters SLC25A12, SLC25A13, SLC25A15, SLC25A18, SLC25A22, and SLC25A29. The widely expressed SLC25A2 transported ADMA across the liposomal membrane in both directions by both unidirectional transport and exchange against arginine or lysine. The SLC25A2-mediated ADMA transport followed first-order kinetics, was nearly as fast as the transport of the best SLC25A2 substrates known so far, and was highly specific as symmetric dimethylarginine (SDMA) was not transported at all. Furthermore, ADMA inhibited SLC25A2 activity with an inhibition constant of 0.38 ± 0.04 mM, whereas SDMA inhibited it poorly. We propose that a major function of SLC25A2 is to export ADMA from mitochondria missing the mitochondrial ADMA-metabolizing enzyme AGXT2. There is evidence that ADMA can also be imported into mitochondria, e.g., in kidney proximal tubulus cells, to be metabolized by AGXT2. SLC25A2 may also mediate this transport function

Expression of solute carrier 7A4 (SLC7A4) in the plasma membrane is not sufficient to mediate amino acid transport activity.

Member 4 of human solute carrier family 7 (SLC7A4) exhibits significant sequence homology with the SLC7 subfamily of human cationic amino acid transporters (hCATs) [Sperandeo, Borsani, Incerti, Zollo, Rossi, Zuffardi, Castaldo, Taglialatela, Andria and Sebastio (1998) Genomics 49, 230-236]. It is therefore often referred to as hCAT-4 even though no convincing transport activity has been shown for this protein. We expressed SLC7A4 in Xenopus laevis oocytes, but could not detect any transport activity for cationic, neutral or anionic amino acids or for the polyamine putrescine. In addition, human glioblastoma cells stably overexpressing a fusion protein between SLC7A4 and the enhanced green fluorescent protein (EGFP) did not exhibit an increased transport activity for l-arginine. The lack of transport activity was not due to a lack of SLC7A4 protein expression in the plasma membrane, as in both cell types SLC7A4-EGFP exhibited a similar subcellular localization and level of protein expression as functional hCAT-EGFP proteins. The expression of SLC7A4 can be induced in NT2 teratocarcinoma cells by treatment with retinoic acid. However, also for this endogenously expressed SLC7A4, we could not detect any transport activity for l-arginine. Our data demonstrate that the expression of SLC7A4 in the plasma membrane is not sufficient to induce an amino acid transport activity in X. laevis oocytes or human cells. Therefore, SLC7A4 is either not an amino acid transporter or it needs additional (protein) factor(s) to be functional