Hydrolysis of Substance P in the Presence of the Osteosarcoma Cell Line SaOS-2: Release of Free Amino Acids

The possible hydrolysis of substance P (Arg–Pro–Lys–Pro–Gln–Gln–Phe–Phe–Gly–Leu–Met) in presence of the osteoblastic cell line SaOS-2 was measured by capillary electrophoresis coupled to mass detection. The results obtained indicate that a very rapid disappearance of the intact undecapeptide was associated to a slower appearance of seven of its eight component amino acids. These results can be interpreted as indicating that an extremely fast hydrolysis of substance P by endopeptidases, which released peptidic by-products, was followed by a noticeably slower secondary degradation which released free amino acids. In decreasing quantitative importance, these phenomena appear to originate by the hydrolysis of the Pro4–Gln5 bond, followed by C-terminal sequential degradation of the Arg1–Pro4 tetrapeptide; by the hydrolysis of or Phe7–Phe8 bond (or, possibly, of Gln6–Phe7) leading to release of free Phe and Gln; by hydrolysis of the Gly9–Leu10 bond with subsequent release of Met and Leu. Results obtained appear to be compatible with the expression by SaOS-2 cells of enzymes already known to catalyze substance P hydrolysis, together with an apparent low efficiency of aminopeptidases. Because of the activity of C-terminal fragments on NK1 receptors, the delay between primary hydrolysis of substance P and secondary hydrolysis of its peptidic fragments indicated by the data shown implies a possible persistence of substance P physiological effects even after degradation of the intact peptide.