Vascular and endothelial actions of inhibitors of substance P amidation

Formation of mature active neuropeptides such as substance P (SP) from thei r glycine extended precursors entails alpha-amidation of peptide precursors by the sequential enzymatic action of peptidylglycine alpha-monooxygenase (PAM) and peptidylamidoglycolate lyase (PGL). We reported that these two en zymes that can produce mature active neuropeptides are present in cultured bovine aortic endothelial cells (BAECs). We hypothesize that alpha-amidatio n of peptides occurs in endothelial cells and that these peptides are criti cally involved in the overall regulation of cardiovascular function. In thi s study, this hypothesis was tested using specific amidation inhibitors to determine their effects on the actions of SP and its glycine-extended precu rsor (SP-Gly). We have found that SP and SP-Gly are equipotent in stimulati ng nitric oxide (NO) release by BAECs. At 10(-5) M, the specific inhibitors of PAM (4-phenyl-3-butenoic acid; PEA) and PGL (5-acetamido-2,4-diketo-6-p henyl-hexanoic acid and its methyl ester) reduced NO basal release by 40, 3 4, and 45%, respectively. They also reduced the production of NO induced by SP-Gly by 63, 68, and 69%, respectively, but had no effect on NO productio n in response to either SP or acetylcholine. SP and SP-Gly also were equipo tent in relaxing rat aortic segments. The vasorelaxation to SP-Gly was endo thelium dependent and inhibited by the NOS antagonist L-nitroarginine methy l ester (L-NAME), but it was not affected by inhibition of prostaglandin sy nthesis. Inhibitors of both PAM and PGL significantly reduced the vasorelax ing actions of SP-Gly, whereas responses to SP were not affected. A cumulat ive infusion of PEA into the femoral artery of rabbits, at final concentrat ions of 2.4, 24, and 240 mu M for 20 min each, increased the vascular resis tance (VR), indicating the tonic production of vasodilating amidated peptid e(s). This effect was maximum at 60 min after infusion (20.5 +/- 4.7 vs. 8. 2 +/- 0.7 mm Hg/ml/min; p < 0.05). These results suggest that endothelial c ells can produce mature SP from its SP-Gly precursor and that a product of peptide alpha-amidation tonically stimulates endothelial cell NO release to control vascular tone.