Signal transduction in CHO cells stably transfected with domain-selective forms of murine ACE.

Membrane-bound human angiotensin-converting enzyme (ACE) has been reported to initiate intracellular signaling after interaction with substrates or inhibitors. Somatic ACE is known to contain two distinct, extracellular catalytic centers. We analyzed the signal transduction mechanisms in cells transfected with different forms of murine ACE (mACE) and investigated whether the two domains are similarly involved in these processes. For this purpose, CHO cells were stably transfected with mACE or with its domain-selective mutants. In addition to these modified cellular models, human umbilical vein endothelial cells were used in this study. Signal transduction molecules such as JNK and c-Jun were analyzed after activation of cells with several ACE substrates and inhibitors. ACE-targeting compounds such as substrates, inhibitors, or even the ACE product angiotensin-II induce in mACE-expressing cells a signal transduction response. These processes are also evoked by partially inactivated forms of mACE and finally result in an enhanced cyclooxygenase-2 transcription. Surprisingly, the membrane-bound ACE activity is also influenced by ACE-targeted interventions. Our data suggest that the two catalytic domains of mACE do not function independently but that the signal transduction is influenced by negative cooperativity of the two catalytic domains. This study underlines that ACE indeed has receptor-like properties which occur in a species-specific manner.

The virtually mature B-type natriuretic peptide (BNP1-32) is a precursor for the more effective BNP1-30.

BACKGROUND AND PURPOSE: The B-type natriuretic peptide (BNP1-32) exerts vasorelaxing and cardioprotective activity. BNP is used as a biomarker for the diagnosis of cardiopathological conditions and recombinant BNP1-32 as a drug for the treatment of such. BNP1-32 has a short half-life and thus, similar to other vasoactive peptides like angiotensin II and bradykinin, can be enzymatically truncated forming bioactive metabolites. We aimed to investigate the metabolism of BNP1-32 in the mouse lung, to identify potential new BNP metabolites and to disclose their biological activity compared to the BNP1-32, in vitro and in vivo. EXPERIMENTAL APPROACH: Using HPLC and MS, we identified a new BNP metabolite, BNP1-30, in the lung being generated by endothelin-converting enzyme-1. KEY RESULTS: BNP1-30 is more efficient in stimulating the guanylyl cyclase (GC) receptor A and, in contrast to BNP1-32, is also able to profoundly stimulate the GC-B. In vivo, BNP1-30 reduced the mean arterial BP of normotensive mice after acute infusion significantly more than BNP1-32. In a model of severe hypertension, a 3-day infusion of BNP1-30 was able to reduce systolic BP by 30 mmHg and to improve markers of heart failure, while BNP1-32 was without significant effect. CONCLUSIONS AND IMPLICATIONS: Our results suggest that BNP1-32 is the precursor for the biologically more active BNP1-30 leading to a fundamental extension of the natriuretic peptide system. Due to expanded activity, BNP1-30 might be a promising treatment option for cardiovascular diseases. Furthermore, its potency as a new diagnostic marker of specific cardiac diseases should be evaluated.

Successive action of meprin A and neprilysin catabolizes B-type natriuretic peptide.

Natriuretic peptides such as B-type natriuretic peptide (BNP) are important cardioprotective hormones with essential functions in sodium excretion, water balance and blood pressure regulation. Consequently, the catabolism of these peptides is in the focus of clinical research. In previous studies, we demonstrated that BNP, in contrast to the structurally related atrial and C-type natriuretic peptide, was not hydrolyzed by neprilysin (NEP). Because membrane preparations of several organs of NEP-knockout mice rapidly degrade BNP, the aim of this study was to identify BNP-catabolizing peptidases responsible for this fast clearance. Using kidney membranes of wild-type and NEP-knockout mice, as well as several peptidase inhibitors, we monitored the catabolism of BNP and analyzed its degradation products. We identified meprin A, a multimeric metalloprotease expressed in the brush borders of kidney proximal tubules, to initially truncate mouse BNP in the N terminus to mBNP7-32, a BNP metabolite with conserved biological activity. Consequently, in vivo experiments with the meprin inhibitor actinonin successfully elevated plasma BNP concentration in rats. We further demonstrated that the generation of mBNP7-32 is the prerequisite to catabolize BNP and identified NEP as the peptidase degrading the truncated BNP. Thus, the cooperative, successive action of the 2 transmembranal peptidases meprin A and NEP is crucial for rapid renal BNP inactivation. Therefore, the inhibition of meprin A could be a potent tool for increasing circulating BNP levels.

Catabolic attacks of membrane-bound angiotensin-converting enzyme on the N-terminal part of species-specific amyloid-beta peptides.

Catabolic processes play a crucial role in the steady state of the amyloid-beta peptide (Abeta). Neprilysin (NEP) and angiotensin-converting enzyme (ACE), two transmembranal enzymes with greatest importance in peptide pharmacology, are known to play a role in Abeta catabolism. This paper focuses on the N-terminal part of Abeta. This region contains the three amino acid residues that determine the differences between human (hAbeta) and murine Abeta (mAbeta). Moreover, the N-terminal part of Abeta contains the zinc-binding site of the molecule. Consequently, all hydrolytic attacks on this part of the Alzheimer peptide should be of exceptional interest. We investigated domain-selective forms of ACE in HPLC-monitored peptide degradation studies and used mass spectrometry for product analyses. We found that ACE-evoked a hydrolysis of the N-terminal part of m- and hAbeta. The hAbeta sequence hAbeta (4-15) was found to be a better substrate for ACE compared to the corresponding murine form. Moreover, we localized the corresponding cleavage sites in the N-terminal part of Abeta as well as in the full-length molecule and identified new sites of endopeptidolytic attack by ACE. Finally, we demonstrate that both catalytic domains of mACE have similar hydrolytic activity on the N-terminal part of Abeta. Our results show that ACE besides its typical function as a dipeptidyl-carboxypeptidase has also unequivocal endopeptidolytic activities.

Central angiotensin II controls alcohol consumption via its AT1 receptor.

Pharmacological and genetic manipulations of the renin-angiotensin system (RAS) have been found to alter the voluntary consumption of alcohol. Here we characterize the role of central angiotensin II (Ang II) in alcohol intake first by using transgenic rats that express an antisense RNA against angiotensinogen and consequently have reduced Ang II levels exclusively in the central nervous system [TGR(ASrAOGEN)680]. These rats consumed markedly less alcohol in comparison to their wild-type controls. Second, Spirapril, an inhibitor of the angiotensin-converting enzyme (ACE), which passes the blood-brain barrier, did not influence the alcohol consumption in the TGR(ASrAOGEN)680, but it significantly reduced alcohol intake in wild-type rats. Studies in knockout mice indicated that the central effect of Ang II on alcohol consumption is mediated by the angiotensin receptor AT1 whereas the AT2 receptor and the bradykinin B2 receptor are not involved. Furthermore, the dopamine concentration in the ventral tegmental area (VTA) is markedly reduced in rats with low central Ang II, strengthening our hypothesis of a role of dopaminergic transmission in Ang II-controlled alcohol preference. Our results indicate that a distinct drug-mediated control of the central RAS could be a promising therapy for alcohol disease.

Structural substrate conditions required for neutral endopeptidase-mediated natriuretic Peptide degradation.

Natriuretic peptides are cyclic vasoactive peptide hormones with great diagnostic and therapeutic relevance. The main catabolic pathway postulated for natriuretic peptides is the degradation by neutral endopeptidase (NEP). However, B-type natriuretic peptide has been found to be resistant to NEP. Here, we compared the degradation of various mature, truncated, and recombinant natriuretic peptides by NEP. The degradation was clearly dependent on the length of the N- or C-terminus as well as on distinct sequence differences within the essential loop structure of the natriuretic peptides. Based on these findings, we developed a model for the interaction of NEP and natriuretic peptides that enables new insights into the mode of action and prediction of substrates of NEP, a peptidase that plays a key role in crucial (patho-) physiological processes.

Interaction of angiotensin-converting enzyme (ACE) with membrane-bound carboxypeptidase M (CPM) - a new function of ACE.

Angiotensin-converting enzyme (ACE) demonstrates, besides its typical dipeptidyl-carboxypeptidase activity, several unusual functions. Here, we demonstrate with molecular, biochemical, and cellular techniques that the somatic wild-type murine ACE (mACE), stably transfected in Chinese Hamster Ovary (CHO) or Madin-Darby Canine Kidney (MDCK) cells, interacts with endogenous membranal co-localized carboxypeptidase M (CPM). CPM belongs to the group of glycosylphosphatidylinositol (GPI)-anchored proteins. Here we report that ACE, completely independent of its known dipeptidase activities, has GPI-targeted properties. Our results indicate that the spatial proximity between mACE and the endogenous CPM enables an ACE-evoked release of CPM. These results are discussed with respect to the recently proposed GPI-ase activity and function of sperm-bound ACE.

Biochemical analysis of neutral endopeptidase activity reveals independent catabolism of atrial and brain natriuretic peptide.

Recent reports presented contradictory results regarding the catabolism of mature atrial (ANP) and brain (BNP) natriuretic peptides in circulation. Especially the role of neutral endopeptidase (NEP) in BNP degradation was conversely discussed. Our present in vitro-studies characterize the NEP-dependent metabolism of ANP and BNP in different tissues via HPLC-analysis using NEP-deficient mice and specific NEP inhibitors. Our results show a strong tissue-dependent degradation pattern of both peptides, which are not only due to the different NEP activities in these tissues. Whereas NEP rapidly degraded ANP, it had no influence in BNP-metabolism. Additional experiments with purified NEP confirmed this result. Moreover, we describe a degradation of ANP and BNP in NEP-deficient- and NEP-inhibited membranes. Consequently, we postulate the existence of at least one further natriuretic peptide (NP) degrading enzyme, which has not been characterized yet. Thus, the commonly accepted model of the natriuretic peptide system with NEP as the central degrading peptidase has to be partly revised. Moreover, the NEP-independent BNP degradation provides an effective means for achieving a beneficial BNP increase in cardiovascular pathology by inhibiting the assumed novel NP-degrading peptidase(s).

Relation of ANP and BNP to their N-terminal fragments in fetal circulation: evidence for enhanced neutral endopeptidase activity and resistance of BNP to neutral endopeptidase in the fetus.

OBJECTIVES: To investigate the role of neutral endopeptidase in the turnover of atrial (ANP) and brain (BNP) natriuretic peptides and their N-terminal fragments in human fetal circulation. DESIGN: Retrospective case-control study. SETTING: Department of Obstetrics and Gynaecology, University of Leipzig, Germany. SAMPLE: Nine control pregnancies and nine pregnancies with rhesus isoimmunisation before and after intravascular transfusion. METHODS: Natriuretic peptides and N-terminal fragments in maternal and fetal blood were measured by radio-immunoassay. Neutral endopeptidase activity was determined by HPLC. MAIN OUTCOME MEASURES: Maternal and fetal plasma concentrations of ANP, NT-proANP, BNP, NT-proBNP as well as neutral endopeptidase activity. Ratios between mature peptide and N-terminal fragment. Feto-maternal ratio. RESULTS: Plasma NT-proANP concentrations are 11.7 times higher in fetal than in maternal circulation. The ANP concentration is only 1.8 times higher, probably due to doubled neutral endopeptidase activity. In contrast, both NT-proBNP and BNP are doubled in fetal plasma. Fetuses with Rh isoimmunisation had significantly higher NT-proBNP but not NT-proANP and neutral endopeptidase activity than controls. An additional volume load by intravascular transfusion did not influence N-terminal fragments or neutral endopeptidase activity. CONCLUSIONS: Our study is the first to determine NT-pro natriuretic peptide concentrations and neutral endopeptidase activity in human fetuses. The results show that increased fetal neutral endopeptidase activity shifts the ANP/NT-proANP but not the BNP/NT-proBNP ratio and that the shifted BNP/NT-proBNP ratio in fetuses with Rh isoimmunisation does not involve increased neutral endopeptidase activity. These findings point to a BNP degradation that is not dependent on neutral endopeptidase.