Interaction of N-carbamoylglutamate, an orphan drug for the treatment of N-acetylglutamate deficiency with glutamate transporters of the SLC1 genfamily

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Disturbances in urea metabolism are associated with hyperammonia, i.e. an increased plasma level of the ammonium ion (NH4+), leading to severe neurological symptoms if not treated properly. Besides restriction of dietary protein and hemodialysis, the orphan drug, N-carbamoylglutamate (NCG) or Carbaglu (trade name), has been approved to treat certain forms of hyperammonemia. NCG activates carbamoylphosphate synthase 1 (CPS-1), the key enzyme of urea synthesis. Physiologically, CPS-1 requires the allosteric activator N-acetylglutamate (NAG) that, in turn, is formed by the N-acetylglutamate synthase (NAGS). Inborn defects in NAGS severely impair urea synthesis. By activating CPS-1, the orally available NCG reduces plasma ammonium close to normal levels in these patients. Previously, we have shown that renal secretion of NCG is accomplished by the sodium-dependent dicarboxylate transporter 3 and the organic anion transporters 1 and 4. The intestinal absorption and hepatic uptake of NCG, however, remained ill-defined. The aim of this proposal is to identify transporters for NCG in small intestine and liver. We shall focus on sodium-dependent glutamate transporters belonging to the SLC-1 family, because NCG can be envisaged as a structural analog of glutamate and these transporters are expressed both in intestinal epithelium and hepatocytes. To this end Xenopus laevis oocytes will be injected with RNA coding for the respective high-affinity glutamate transporters SLC1A1, 2, and 3. After expression of the proteins in the plasma membrane, NCG-mediated currents can be detected and set in relation to glutamate-evoked currents. Additional uptake experiments using radio-labeled glutamate in the absence and presence of NCG and transporter-specific inhibitors in human enterocytes and hepatocytes supported by antibody studies will help to identify the transporters responsible for the uptake of NCG in enterocytes and hepatocytes.