engleski

Functional properties of a novel peptide transporter in undifferentiated PC12 (rat neuroendocrine) cell line

It is well recognized that small peptides are absorbed into epithelial cells by one of the two members; PepT1 or PepT2, of the H+/oligopeptide transporter (POT) family (reviewed in Meredith, & Boyd, 2000). Peptide transport in neurons or endocrine cells is less well studied. A rat pheochromocytoma cell line (PC12) has therefore been used as a model to explore and screen for endogenous peptide transporters. The cells were grown in 12-well plates at 500 000 cells/well in complete medium (RPMI 1640 medium supplemented with 10 % heat inactivated horse serum, 5 % fetal bovine serum, 4 mM glutamine and 175 ug/ml gentamycin) at 370C in a 5% CO2 and 95 % air environment (Greene et al. 1997). Prior to uptake experiments, cells were washed and equilibrated (15-30 min, 37 0C ) in HEPES buffered Krebs pH 7.4. The initial rate of uptake of the hydrolysis-resistant anionic peptide [3H]-D-Phe-L-Glu (420 nM, specific activity 10 Ci/mmole) was determined at 370C in the absence or presence of potential substrates for PepT1 and PepT2. All experiments were performed at pH 5.5 (MES-buffered in the presence of any inhibitory peptides used) unless otherwise stated. Non-specific uptake (i.e. 0 sec) was subtracted from all data points. In control PC12 cells, there was time-dependent uptake of labelled D-Phe-L-Glu. Surprisingly, the uptake was scarcely inhibited by the addition of 5 mM unlabelled D-Phe-L-Glu or L-Phe-L-Val. Interestingly, the uptake of labelled D-Phe-L-Glu was strongly inhibited (85.7 ą 1.8 % of control, mean ą S.E.M n=3) by 5 mM L-Arg-L-Ala, whereas the constituent amino acids L-Arg plus L-Ala (5 mM) were very much less effective. The basic dipeptides L-Arg-L-Gly and L-Lys-L-Ala caused similar inhibition. The Ki for L-Arg-L-Ala inhibition of [3H]-D-Phe-L-Glu uptake was 0.6 ą 0.1 mM, (mean ą S.E.M n=3). The uptake of labelled dipeptide was also examined in cells which were depolarised by raised external potassium concentration (45 mM KCl isotonically replacing NaCl at pH 5.5 and 7.4) in the presence or absence of 5mM unlabelled L-Arg-L-Ala. These experiments suggest that the transport system is not electrogenic since altered membrane potential causes little effect on [3H]-D-Phe-L-Glu uptake at acidic external pH. This preliminary functional data suggests a novel peptide transporter in PC12 cells.

peptide transporter; undifferentiated PC12

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