Fig. 8. Trans-stimulation of PAT2-mediated l-[3H]proline efflux from oocytes discriminates between PAT2 substrates and non-transported inhibitors. PAT2-expressing and water-injected oocytes pre-loaded with l-[3H]proline (approximately 4 mM) were incubated (10 min) in pH 5.5 Na+-free solution in the presence or absence of various amino acids (all 10 mM). A. A typical trans-stimulation experiment to illustrate efflux of l-[3H]proline from PAT2-expressing (filled columns) and water-injected (open columns) oocytes in the absence (control) or presence of glycine (Gly), 5-hydroxy-l-tryptophan (OH-Trp) or α-methyl-d,l-tryptophan (Me-Trp) (all 10 mM). Data are mean ± SEM (n = 10). ***, p < 0.001; ns, p > 0.05, versus water-injected oocytes. B. l-[3H]Proline efflux was measured in the absence (control) or presence of glycine (Gly), l-alanine (Ala), sarcosine (Sar), glutamic acid (Glu), isoguvacine (Isoguv), isonipecotic acid (Isonip), guvacine (Guv), l-pipecolic acid (l-Pip), 5-hydroxy-l-tryptophan (OH-Trp) and α-methyl-d,l-tryptophan (Me-Trp) (all 10 mM). Results are expressed as the percentage of l-[3H]proline efflux in the presence of 10 mM extracellular glycine. Data are mean ± SEM (n = 5–10). ***, p < 0.001; ns, p > 0.05, versus efflux under control conditions (efflux from water-injected oocytes under identical conditions). C. Relationship between the abilities of various amino acids and derivatives to cis-inhibit PAT2-mediated amino acid uptake and trans-stimulate PAT2-mediated amino acid efflux. For cis-inhibition, l-[3H]proline (10 μM) uptake (pH 5.5, Na+-free) was measured in PAT2-expressing oocytes in the absence and presence of unlabelled amino acids (all 10 mM). Results were determined as PAT2-specific uptake (following subtraction of uptake in water-injected oocytes under identical experimental conditions) and are expressed as a percent of the inhibition observed in the presence of 10 mM glycine. Data are mean ± SEM (n = 20). For trans-stimulation, data are taken from panel B, and are expressed as the percent of the trans-stimulation observed in the presence of 10 mM glycine. The relationship [excluding l-pipecolic acid (l-Pip), 5-hydroxy-l-tryptophan (OH-Trp) and α-methyl-d,l-tryptophan (Me-Trp)] was analysed by linear regression (r2 = 0.82).
Image published in: Edwards N et al. (2011)
© 2011 Elsevier B. This image is reproduced with permission of the journal and the copyright holder. This is an open-access article distributed under the terms of the Creative Commons Attribution license
Permanent Image Page
Printer Friendly View
XB-IMG-125555