The relative roles of phenylalanine and tyrosine as substrates for DOPA synthesis in PC12 cells.

The relative contributions of tyrosine (TYR) and phenylalanine (PHE) to the synthesis of dihydroxyphenylalanine (DOPA) were studied in PC12 cells following inhibition of aromatic L-amino acid decarboxylase with m-hydroxybenzylhydrazine (NSD-1015). Cells were incubated with varying concentrations of unlabeled L-TYR and L-PHE, and either L-(3)H-TYR or L-(3)H-PHE. Following incubation, labeled and unlabeled TYR, PHE, and DOPA were quantitated following HPLC separation. PC12 cells synthesized DOPA from both TYR and PHE. Raising the concentration of one amino acid relative to that of the other increased the proportion of DOPA synthesized from that amino acid. TYR suppressed DOPA synthesis from (3)H-PHE at concentrations lower than that observed for a similar inhibition by PHE of DOPA synthesis from (3)H-TYR. Inhibition of total DOPA synthesis occurred only at high concentrations of either amino acid. The results suggest that in the PC12 cell, TYR and PHE can be used interchangeably as substrates for TYR hydroxylation, and that the proportion of catecholamine synthesized will depend on the relative proportions of each substrate available to the cell. However, TYR is clearly the preferred substrate for tyrosine hydroxylase.

The effect of phenylalanine on DOPA synthesis in PC12 cells.

DOPA synthesis from phenylalanine was studied in PC12 cells incubated with m-hydroxybenzylhydrazine, to inhibit aromatic L-amino acid decarboxylase. DOPA synthesis rose with increasing concentrations of either phenylalanine or tyrosine; maximal rates (approximately 55 pmol/min/mg protein for tyrosine; approximately 40 pmol/min/mg protein for phenylalanine) occurred at a medium concentration of approximately 10 microM for either amino acid. The Km for either amino acid was about 1 microM (medium concentration). At tyrosine concentrations above 30 microM, DOPA synthesis declined; inhibition was observed at higher concentrations for phenylalanine (> or =300 microM). These effects were most notable in the presence of 56 mM potassium. Measurements of intracellular phenylalanine and tyrosine suggested the Km for either amino acid is 20-30 microM; maximal synthesis occurred at 120-140 microM. In the presence of both phenylalanine and tyrosine, DOPA synthesis was inhibited by phenylalanine only at a high medium concentration (1000 microM), regardless of medium tyrosine concentration. The inhibition of DOPA synthesis by high medium tyrosine concentrations was antagonized by high medium phenylalanine concentrations (100, 1000 microM). Together, the findings indicate that for PC12 cells, phenylalanine can be a significant substrate for tyrosine hydroxylase, is a relatively weak inhibitor of the enzyme, and at high concentrations can antagonize substrate inhibition by tyrosine.

Effects of membrane fluidity on [3H]TCP binding to PCP receptors.

Phencyclidine (PCP) binds with high affinity to the ion channel associated with the NMDA receptor. The binding of the PCP receptor-specific ligand TCP is greatly reduced at temperatures between 2 degrees C and 6 degrees C, at which the plasma membrane is in a rigid state. However, membrane rigidity alone does not appear to cause the reduced TCP binding, since the membrane fluidizing agent A2C did not increase TCP binding at 4 degrees C; instead, it decreased binding at 21 degrees C. This inhibitory effect of A2C on TCP binding was dose dependent and was highly correlated with A2C-induced increases in membrane fluidity. The IC50 of A2C inhibition was 8.9 mM, with a pseudo-Hill coefficient of -0.24. Scatchard analysis demonstrated that this effect was the result of an increase in the apparent KD of [3H]TCP for the PCP receptor, with no effect on the Bmax. These results suggest that the function of the NMDA-PCP receptor complex is impaired by increases in membrane fluidity. These findings may be pharmacologically relevant in understanding the mechanism of action of such agents as general anesthetics and ethanol, which cause increases in plasma membrane fluidity.