Effects of the N-Terminal Sequence of ACE on the Properties of Its C-Domain

Abstract

Abstract-Angiotensin I-converting enzyme (ACE, kininase II) has 2 active domains (N and C) in a single peptide chain.Because we found its N-domain more stable than its C-domain, we investigated the effect of the amino-terminus of human ACE on the C-domain with a molecular construct expressed in Chinese hamster ovary cells (CHO) cells and transiently in HEK293 cells. This active N-deleted ACE contained only the first 141 amino acids of the human N-domain but not its active center and was linked to the active C-domain containing the transmembrane and cytosolic portions of ACE. The CHO cells were also transfected with human B 2 bradykinin receptor. ACE inhibitors (5 nmol/L or 1 mol/L) augmented bradykinin (100 nmol/L) effects, elevated B 2 receptor numbers, and resensitized the receptor desensitized by agonist as measured by arachidonic acid release or [Ca 2ϩ ] i mobilization. Arachidonic acid release was mediated by pertussis toxin-sensitive G␣ i , and [Ca 2ϩ ] i mobilization was mediated by pertussis-insensitive G␣ q protein receptor complex. The properties of the construct were compared with wild-type ACE and separate N-and C-domains. The N-deleted ACE differed from wild-type in activation by Cl Ϫ and [SO 4 ] 2Ϫ ions, hydrolysis ratios of substrates (both short synthetic and endogenous peptides) and heat stability. Thus, the N-terminal peptide of ACE affected the characteristics of the C-domain active center. ACE inhibitors acting on N-deleted ACE, which had only a single C-domain active center anchored to plasma membrane, induced cross-talk between the enzyme and the B 2 receptor (eg, the inhibitors resensitized the receptor) independent of blocking bradykinin inactivation.

Kallikrein activates bradykinin B2 receptors in absence of kininogen

“…To obtain CHO cells co-expressing both human ACE and bradykinin B 2 receptor, the cells were first transfected with human ACE pcDNA6 plasmid, and individual clones were screened for ACE activity (11,34). Clones with high ACE activity were then transfected with human BK B 2 receptor cDNA as described above (CHO AB).…”

“…Thus, the rat and hamster livers were strongly positive, showing appreciable amounts of mRNA, but cultured CHO cells lacked kininogen mRNA, confirming the results of our experiments (23,25) that kallikrein indeed activates the B 2 BK receptors without releasing kinin. (15,(33)(34)(35) by a mechanism independent of blocking BK inactivation. To determine how ACE inhibitors potentiate kinin receptor agonists, we stably transfected CHO cells bearing human B 2 BK receptors with human ACE and adapted them to grow in serum-free medium (CHO AB/PFAF cells).…”

“…Kinins release potent vasodilators, such as prostaglandins (PG), nitric oxide (NO), and endothelial-derived hyperpolarizing factor (EDHF) (3)(4)(5), which influence blood pressure and vessel tone, but they can cause pain and enhance capillary permeability in inflammation as well (3). The beneficial effects of angiotensin I-converting enzyme (ACE) (18,40), or kininase II are attributed at least in part to prolongation of the half-life of bradykinin (BK) (6)(7)(8)(20)(21)(22) and potentiation of its effects on the B 2 receptor (15,(33)(34)(35). Although plasma kininogen is the primary substrate of kallikreins, these serine proteases can cleave other proteins as well, such as Factor XII, the activator of prokallikrein (12,41,42).…”

“…We also investigated the effects of ACE inhibitors, because these agents appear to potentiate kinin effects through crosstalk between ACE and B 2 receptors (15,(33)(34)(35). The results from these experiments enabled us to exclude kinin release as a mechanism for kallikrein activation of B 2 receptors.…”

Ace inhibitors - activators of kinin receptors

“…Полученные данные привели к предположению, что активация Б 2 Р, наблюдаемая в присутствии молекул АПФ, по-видимому, связана с индукцией иАПФ перекрестного взаимодействия между АПФ и Б 2 Р, находящимися на мембране. Другим исследователям удалось доказать существование физического взаимодействия молекул АПФ и Б 2 Р на клеточной мембране [15,81,82]. Было установлено, что для активации Б 2 Р иАПФ не требуются ни цитоплазматический, ни трансмембранный фрагменты АПФ; активация происходит, если АПФ находится в непосредственной близости от рецептора, что, возможно, приводит к образованию гетеродимера за счет внеклеточных фрагментов молекул АПФ и Б 2 Р. Близкие результаты были получены при использовании двух ингибиторов АПФ -эналаприла и рамиприла, что указывает на группоспецифическое действие этих соединений на активацию Б 2 Р, которое, по-видимому, не связано с их индивидуальной структурой.…”