Bachelard Hélène, Charest-Morin Xavier, Marceau François
Division of Endocrinology and Nephrology, Centre Hospitalier Universitaire de Québec Research Center-CHUL, Laval University, Quebec, QC, Canada.
Division of Infectious Diseases and Immunity, Centre Hospitalier Universitaire de Québec Research Center-CHUL, Laval University, Quebec, QC, Canada.
Front Pharmacol. 2018 Mar 27;9:273. doi: 10.3389/fphar.2018.00273. eCollection 2018.
We previously reported hypotensive and vasodilator effects from C-terminally extended bradykinin (BK) sequences that behave as B receptor (BR) agonists activated by vascular or plasma peptidases. D-Arg-BK-Arg-Arg (r-BK-RR) is a novel prodrug peptide hypothetically activated by two catalytic cycles of Arg-carboxypeptidases (CPs) to release the direct agonist D-Arg-BK. N-terminally extending the BK sequence with D-Arg in the latter peptide was meant to block the second kinin inactivation pathway in importance, aminopeptidase P. The affinity of r-BK and r-BK-RR for recombinant BR was assessed using a [H]BK binding displacement assay. Their pharmacology was evaluated in human isolated umbilical vein, a contractile bioassay for the BR, in a morphological assay involving the endocytosis of BR-green fusion protein (GFP) and in anesthetized rats instrumented to record hemodynamic responses to bolus intravenous injection of both peptides. r-BK exhibited an affinity equal to that of BK for the rat BR, while r-BK-RR was 61-fold less potent. In the vein and the BR-GFP internalization assay, r-BK was a direct agonist unaffected by the blockade of angiotensin converting enzyme (ACE) with enalaprilat, or Arg-CPs with Plummer's inhibitor. However, the effects of r-BK-RR were reduced by these inhibitors, more so by enalaprilat. In anesthetized rats, r-BK and r-BK-RR were equipotent hypotensive agents and their effects were inhibited by icatibant (a BR antagonist). The hypotensive effects of r-BK were potentiated by enalaprilat, but not influenced by the Arg-CPs inhibitor, which is consistent with a minor role of Arg-CPs in the metabolism of r-BK. However, in rats pretreated with both enalaprilat and Plummer's inhibitor, the hypotensive responses and the duration of the hypotensive episode to r-BK were significantly potentiated. The hypotensive responses to r-BK-RR were not affected by enalaprilat, but were reduced by pre-treatment with the Arg-CPs inhibitor alone or combined with enalaprilat. Therefore, , Arg-CPs activity is dominant over ACE to regenerate the BR agonist r-BK from r-BK-RR, a prodrug activator of the BR. A BR agonist activated only at the level of the microcirculation by resident peptidases could be developed as an intravenously infused drug for ischemic diseases.
我们之前报道过，C末端延长的缓激肽（BK）序列具有降压和血管舒张作用，其作为血管或血浆肽酶激活的B受体（BR）激动剂。D-Arg-BK-Arg-Arg（r-BK-RR）是一种新型前药肽，推测可通过精氨酸羧肽酶（CPs）的两个催化循环激活，以释放直接激动剂D-Arg-BK。在后一种肽中用D-Arg在N末端延长BK序列旨在阻断重要性次之的第二条激肽失活途径，即氨肽酶P。使用[H]BK结合置换试验评估r-BK和r-BK-RR对重组BR的亲和力。在人离体脐静脉（一种用于BR的收缩生物测定法）、涉及BR-绿色荧光蛋白（GFP）内吞作用的形态学测定法以及用于记录对两种肽静脉推注的血流动力学反应的麻醉大鼠中评估它们的药理学特性。r-BK对大鼠BR的亲和力与BK相当，而r-BK-RR的效力低61倍。在静脉和BR-GFP内化试验中，r-BK是一种直接激动剂，不受依那普利拉对血管紧张素转换酶（ACE）的阻断或普卢默抑制剂对精氨酸羧肽酶的阻断的影响。然而，这些抑制剂可降低r-BK-RR的作用，依那普利拉的作用更明显。在麻醉大鼠中，r-BK和r-BK-RR是等效的降压剂，它们的作用可被艾替班特（一种BR拮抗剂）抑制。r-BK的降压作用可被依那普利拉增强，但不受精氨酸羧肽酶抑制剂的影响，这与精氨酸羧肽酶在r-BK代谢中的次要作用一致。然而，在同时用依那普利拉和普卢默抑制剂预处理的大鼠中，对r-BK的降压反应和降压发作的持续时间显著增强。对r-BK-RR的降压反应不受依那普利拉的影响，但单独用精氨酸羧肽酶抑制剂或与依那普利拉联合预处理可使其降低。因此，精氨酸羧肽酶活性在从r-BK-RR（一种BR的前药激活剂）再生BR激动剂r-BK方面比ACE占主导地位。一种仅在微循环水平被驻留肽酶激活的BR激动剂可被开发为用于缺血性疾病的静脉输注药物。
