Vascular and Endothelial Actions of Inhibitors of Substance P Amidation

Abstract

Formation of mature active neuropeptides such as substance P (SP) from their 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 enzymes that can produce mature active neuropeptides are present in cultured bovine aortic endothelial cells (BAECs). We hypothesize that alpha-amidation of peptides occurs in endothelial cells and that these peptides are critically involved in the overall regulation of cardiovascular function. In this study, this hypothesis was tested using specific amidation inhibitors to determine their effects on the actions of SP and its glycine-extended precursor (SP-Gly). We have found that SP and SP-Gly are equipotent in stimulating nitric oxide (NO) release by BAECs. At 10(-5) M, the specific inhibitors of PAM (4-phenyl-3-butenoic acid; PBA) and PGL (5-acetamido-2,4-diketo-6-phenyl-hexanoic acid and its methyl ester) reduced NO basal release by 40, 34, 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 production in response to either SP or acetylcholine. SP and SP-Gly also were equipotent in relaxing rat aortic segments. The vasorelaxation to SP-Gly was endothelium dependent and inhibited by the NOS antagonist L-nitroarginine methyl ester (L-NAME), but it was not affected by inhibition of prostaglandin synthesis. Inhibitors of both PAM and PGL significantly reduced the vasorelaxing actions of SP-Gly, whereas responses to SP were not affected. A cumulative infusion of PBA into the femoral artery of rabbits, at final concentrations of 2.4, 24, and 240 microM for 20 min each, increased the vascular resistance (VR), indicating the tonic production of vasodilating amidated peptide(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 cells 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.

Inhibition of JNK and p38 MAPK phosphorylation by 5-(acetylamino)-4-oxo-6-phenyl-2-hexenoic acid methyl ester and 4-phenyl-butenoic acid decreases substance P-induced TNF-α upregulation in macrophages

“…It is has long been assumed that the glycine-extended precursors of bioactive peptides have little or no bioactivity prior to amidation [14]. For example, SP-Gly-induced relaxation of rat aortic strips and stimulation of NO production in bovine endothelial cells is markedly reduced in the presence of inhibitors which prevent conversion of SP-Gly to SP [22, 35]. It is therefore apparent that, at least under our experimental conditions, RAW 264.7 macrophages are unable to amidate exogenous SP-Gly to a degree that would stimulate TNF-α production.…”

Divergent roles for ferric ions in the biological activity of amidated and non-amidated gastrins

“…Although C-terminal amidation was thought to be essential for the bioactivity of most peptide hormones, there are an increasing number of examples where a glycine-extended intermediate is also bioactive. Examples include secretin-gly, gastrin releasing peptide (GRP)-gly, glucagon-like peptide (GLP)-1-gly and substance P (Wettergren et al 1998, Abou-Mohamed et al 2000, Oiry et al 2000, Solomon et al 2001. However, these glycine-extended forms either interact with the same receptor type as the amidated form or are converted to the mature form close to the site of action.…”

Endogenous Vasoactive Peptides

“…At least some of substance P's actions result from its ability to suppress the synthesis and release of catecholamines from the adrenal medulla [589], as well as alterations in ACTH-corticosterone production [590]. It has recently been shown that the final a-amidation step necessary for full activity of substance P can occur directly within endothelial cells and can be released by shear stress [591,592]. This endothelialderived substance P can then evoke vasorelaxation through a nitric oxide-dependent mechanism [591].…”

“…It has recently been shown that the final a-amidation step necessary for full activity of substance P can occur directly within endothelial cells and can be released by shear stress [591,592]. This endothelialderived substance P can then evoke vasorelaxation through a nitric oxide-dependent mechanism [591]. Interestingly, the immediate precursor prior to the a-amidation, substance P-Gly, also possesses a vasorelaxant potency similar to that of the mature peptide [593].…”