Galactosyl Derivatives of l-Arginine and d-Arginine:  Synthesis, Stability, Cell Permeation, and Nitric Oxide Production in Pituitary GH3 Cells

Nitric oxide (NO) is critical for the normal physiological regulation of the nervous system and other tissues. l-Arginine, but not d-arginine, is the natural substrate for nitric oxide synthase (NOS), for it is enzymatically converted to NO and l-citrulline. However, recent evidence suggests that d-arginine can also produce NO and NO-derivatives via a different pathway. The aim of the present paper was to raise NO levels in the cells by increasing the cell permeation of its precursors. To this aim, two galactosyl prodrugs, l-arginine-d-galactos-6‘-yl ester (l-ArgGal) and d-arginine-d-galactos-6‘-yl ester (d-ArgGal) were synthesized. Remarkably, using the HPLC-ESI/MS technique, we found that l-ArgGal and d-ArgGal prodrugs both increased the concentration levels of l- and d-arginine and their derivatives in pituitary GH3 cells. Furthermore, we found that d-ArgGal (1) penetrated cell membranes more rapidly than its precursor d-arginine, (2) released arginine more slowly and in greater amounts than l-ArgGal, and (3) produced much higher levels of DAF-2 monitored NO and nitrite than did l-ArgGal under the same experimental conditions. In conclusion, these results indicate that an increase in the cell permeation of l- and d-arginine by l-ArgGal and d-ArgGal can lead to an increase in NO levels.