STRUCTURAL ANALOGS OF PYRROLINE 5-CARBOXYLATE SPECIFICALLY INHIBIT ITS UPTAKE INTO CELLS

Pyrroline 5-carboxylate, a naturally occurring intermediate, is a potent activator of redox-dependent metabolic pathways. This effect of pyrroline 5-carboxylate is due, at least in part, to the special mechanism mediating its entry into cells. Using Chinese hamster ovary cells we recently characterized the cellular uptake of pyrroline 5-carboxylate as a process transferring oxidizing potential pari passu with cell entry, a process consistent with group translocation. We sought to identify specific inhibitors to probe this unique uptake mechanism, to blockade the metabolic effects of pyrroline 5-carboxylate, and to provide strategies to identify the putative carrier protein. Because pyrroline 5-carboxylate, a ring structure with a tertiary nitrogen, is in spontaneous equilibrium with glutamic-gamma-semialdehyde, an open-chain structure, we tested analogues of both. Most open-chain aldehydes at 10 mM had little effect on the uptake of pyrroline 5-carboxylate. Although succinic semialdehyde did inhibit, its effect was nonspecific in that the uptake of alpha(methylamino) isobutyric acid was inhibited as much as the uptake of pyrroline 5-carboxylate. In contrast, pyrroline 2-carboxylate and other cyclic compounds with tertiary nitrogens, e.g., pyridines, were specific inhibitors of pyrroline 5-carboxylate uptake. Respective potencies of pyridine derivatives depended on the nature and location of constituent groups. Kinetics studies showed that these inhibitors were competitive with pyrroline 5-carboxylate and the most potent inhibitor, 2,6-pyridinedicarboxaldehyde, exhibited a K12 of 0.27 +/- 0.05 mM. In the face of their effect on P5C uptake, the most potent of these analogues, 2-pyridinecarboxaldehyde and 2,6-pyridinedicarboxaldehyde, did not inhibit the activity of pyrroline 5-carboxylate reductase, the enzyme that converts pyrroline 5-carboxylate to proline. Nevertheless, the analogues markedly inhibited the stimulatory effect of P5C on the pentose phosphate shunt. Importantly, not only did 2-pyridinecarboxaldehyde protect the pyrroline 5-carboxylate uptake mechanism from the inhibitory effects of a sulfhydryl-reactive agent, but also its inhibitory effect became irreversible in the presence of sodium cyanoborohydride. These inhibitors may help discriminate events mediated by the transport carrier from those mediated by intracellular metabolism and may provide a method for identifying and characterizing the putative carrier for P5C.