Modulation of cellular signaling mechanisms by qualitative differences in dietary proteins and their metabolites were studied at the level of a) effect of pyrroline 5-carboxylate (P5C) on mitogenesis--effect on membrane phosphoinositides, b) P5C as a diet-dependent scavenger of nitric oxide, c) enzymes mediating the formation and degradation of P5C, and d) imidodipeptides. Effect of pyrroline 5-carboxylate on mitogenesis. P5C stimulates PRPP and purine ribonucleotide synthesis synergistically with platelet-derived growth factor. It also increases the incorporation of thymidine in serum- activated cells. Inhibitor studies suggest that the effect is due to the turnover of membrane phosphoinositides and direct assays show that it is phospholipase D which is stimulated by P5C with the release of phosphatidic acid. Possible interaction of this system with various growth factors and its modulation of intermediate metabolism are being pursued. Pyrroline 5-carboxylate as a diet-dependent scavenger of nitric oxide. P5C is a circulating metabolite, and its levels in plasma undergo diet- dependent diurnal fluctuations. We recently found that P5C interacts with nitric oxide to form nitrosoproline. The scavenging of NO by P5C readily occurs at physiologic pH and at concentrations of P5C found in plasma. Biologic regulation of this process is being intensively studied. Enzymes mediating the synthesis and degradation of pyrroline 5- carboxylate. We are studying the enzymes of P5C metabolism at the molecular level. Specifically, ornithine aminotransferase, P5C reductase and P5C dehydrogenase are being studied. Using Northern blots, we are studying the expression of these enzymes in animals and in cultured cells. The regulation of these enzymes has become of great interest since they not only mediate the formation of arginine critical for NO formation but may also regulate P5C as a scavenger of excess P5C. Imidodipeptides. Dipeptides containing proline or hydroxyproline originate from either tissue matrix degradation or from protein nutrition. They circulate in plasma and are delivered to tissues where they are hydrolyzed by prolidase. Thus, prolidase is a potential interface between protein nutrition and matrix breakdown. Our studies showed that the level of cellular prolidase is regulated by extracellular collagen acting through integrin receptors. Thus, the hydrolysis of imidodipeptides, the final degradative products of matrix collagen, is responsive to cellular interaction with extracellular matrix. We are studying the regulation of this enzyme on the molecular level.
