Abou-Mohamed G A, Huang J, Oldham C D, Taylor T A, Jin L, Caldwell R B, May S W, Caldwell R W
Department of Pharmacology & Toxicology, Medical College of Georgia, Augusta 30912, USA.
J Cardiovasc Pharmacol. 2000 Jun;35(6):871-80. doi: 10.1097/00005344-200006000-00007.
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.
从甘氨酸延伸前体形成成熟的活性神经肽，如P物质（SP），需要肽基甘氨酸α-单加氧酶（PAM）和肽基酰胺基乙醇酸裂解酶（PGL）的顺序酶促作用对肽前体进行α-酰胺化。我们报道，这两种能够产生成熟活性神经肽的酶存在于培养的牛主动脉内皮细胞（BAECs）中。我们假设肽的α-酰胺化发生在内皮细胞中，并且这些肽在心血管功能的整体调节中起关键作用。在本研究中，使用特异性酰胺化抑制剂来测试这一假设，以确定它们对SP及其甘氨酸延伸前体（SP-Gly）作用的影响。我们发现SP和SP-Gly在刺激BAECs释放一氧化氮（NO）方面具有同等效力。在10^(-5) M时，PAM的特异性抑制剂（4-苯基-3-丁烯酸；PBA）和PGL的特异性抑制剂（5-乙酰氨基-2,4-二酮-6-苯基己酸及其甲酯）分别使NO基础释放量降低了40%、34%和45%。它们还分别使SP-Gly诱导的NO生成量降低了63%、68%和69%，但对SP或乙酰胆碱诱导的NO生成没有影响。SP和SP-Gly在舒张大鼠主动脉段方面也具有同等效力。对SP-Gly的血管舒张作用是内皮依赖性的，并被一氧化氮合酶拮抗剂L-硝基精氨酸甲酯（L-NAME）抑制，但不受前列腺素合成抑制的影响。PAM和PGL的抑制剂均显著降低了SP-Gly的血管舒张作用，而对SP的反应没有影响。以最终浓度2.4、24和240 microM分别向兔股动脉累积输注PBA 20分钟，增加了血管阻力（VR），表明存在血管舒张性酰胺化肽的持续性产生。这种作用在输注后60分钟时最大（20.5±4.7对8.2±0.7 mmHg/ml/min；p<0.05）。这些结果表明，内皮细胞可以从其SP-Gly前体产生成熟的SP，并且肽α-酰胺化的产物持续性刺激内皮细胞释放NO以控制血管张力。
