Elsevier

Peptides

Volume 26, Issue 8, August 2005, Pages 1317-1322
Peptides

Hypotensive effects of hemopressin and bradykinin in rabbits, rats and mice: A comparative study

https://doi.org/10.1016/j.peptides.2005.03.026Get rights and content

Abstract

Hemopressin is a novel vasoactive nonapeptide derived from hemoglobin's α-chain as recently reported by Rioli et al. [Rioli V, Gozzo FC, Heimann AS, Linardi A, Krieger JE, Shida CS, et al. Novel natural peptide substrates for endopeptidase 24.15, neurolysin, and angiotensin-converting enzyme. J Biol Chem 2003;278(10):8547–55]. In anesthetized male Wistar rats, this peptide exhibited hypotensive actions similar to those of bradykinin (BK) when administered intravenously (i.v.), and was found to be metabolized both in vitro and in vivo by several peptidases, including the angiotensin-converting enzyme (ACE). In this study, these findings were expanded upon by examining: (i) the degradation kinetics following incubation with ACE purified from rabbit lung and (ii) the blood pressure lowering effects of HP and BK injected i.v. or intra-arterially (i.a.) in male rabbits, rats, and mice. Our findings demonstrate that, in vitro, HP and BK are both degraded by ACE, but at different velocity rates. Furthermore, both HP and BK induced transient hypotension in all animals tested, although the responses to HP relative to the administration sites were significantly lower (by 10–100-fold) on an equimolar basis compared to those of BK. In rabbits, the decrease of blood pressure induced by HP (10–100 nmol/kg) did not differ whether it was administered i.v. or i.a., suggesting an absence of pulmonary/cardiac inactivation in contrast to BK (0.1–1 nmol/kg). The in vivo effect of HP was significantly potentiated in rabbits immunostimulated with bacterial lipopolysaccharide (LPS), but was unaffected by both the B2 receptor antagonist HOE 140 (0.1 μmol/kg) and captopril (100 μg/kg), contrary to BK. Therefore, HP acts as a weak hypotensive mediator, which does not activate kinin B2 receptors, but uses a functional site and/or signaling paths appearing to be up-regulated by LPS.

Introduction

In the process of investigating potential endogenous substrates for endopeptidase EC 3.4.24.15 (ep24.15) (alias thimet oligopeptidase) and endopeptidase EC 3.4.24.16 (ep24.16) (alias neurolysin), Rioli et al. [18] observed that one of the peptides isolated from rat brain extract, which binds to ep24.15 and ep24.16 enzymes catalytically inactivated by site-directed mutagenesis, produced a hypotensive response when administered intravenously to rats. This novel vasoactive nonapeptide, hemopressin (HP) (H-PVNFKFLSH-OH), was subsequently found to originate from the α1-chain of hemoglobin. HP is thought to be generated through the degradation of oxidatively modified hemoglobin by the proteasome complex [7], [12], [15], [19], likely during phagocytic scavenging of red blood cells or, to a lesser extent, directly in the circulating erythrocytes. Even less is known about how this short peptide may reach its extracellular target. Moreover, HP belongs to the bourgeoning list of substrates for metalloendopeptidase angiotensin-converting enzyme (ACE; EC 3.4.15.1), ep24.15 and ep24.16 enzymes [18]. ACE is a ubiquitous enzyme expressed prominently in pulmonary microvascular endothelial cells, and plays a fundamental role in cardiovascular homeostasis [1], [20]. Although ep24.15 and ep24.16 enzymes were first described as soluble cytosolic enzymes, recent evidence suggests that they may also be distributed at the plasmalemmal membranes of endothelial cells to inactivate (or activate) extracellular bioactive peptides (e.g. angiotensin I, BK, substance P) [13].

One red blood cell contains 2.7 × 108 molecules of hemoglobin. The adult human body has approximately 2× 1013 to 3 × 1013 red blood cells and, of these, some 3 million die every second and are scavenged by phagocytic cells in the liver and spleen. With hemoglobin being the major pool of HP, and considering the increased potential for its role in pathological conditions where erythrocyte damage occurs (e.g. septicemic hemolysis), we were prompted to revisit some of the findings of Rioli et al. [18] in an attempt to broaden the spectrum of knowledge on the hemodynamic properties of this novel bioactive peptide. The blood pressure altering effects of HP were ascertained in different animal species, namely the rabbit, rat and mouse, and its susceptibility toward proteolytic degradation was measured following in vitro incubation with ACE. We also investigated whether HP in vivo responses are modulated under pathological conditions, specifically septicemia, as reported for other peptidergic receptor systems such as the kinin B1 receptors [2], [11], [17].

Section snippets

Peptide synthesis

Bradykinin (BK), desArg9BK, rabbit/mouse HP isoform (H-PVNFKLLSH-OH) deduced via the SIB BLAST network service, rat HP isoform (rHP) (H-PVNFKFLSH-OH) and its fragments (H-PVNFK-OH, H-PVNFKF-OH, and H-PVNFKFL-OH), as well as HOE 140 (DArg[Hyp3,Thi5,DTic7,Oic8]BK) were synthesized by continuous flow synthesis on a Pioneer Peptide Synthesis System (PerSeptive Biosystems) using the Fmoc strategy. Material and chemicals were obtained from the following sources: NovaSyn TGA resin for arginine,

Analysis of HP and BK peptide processing by ACE

Incubation of BK (Fig. 1A) and rHP (Fig. 1B) with ACE in vitro generated BK 1–7 (m/z 756.4) and rHP 1–7 (m/z 863.4) in agreement with the established dipeptidyl carboxypeptidase activity of the enzyme [20]. A further cleavage of BK 1–7 and rHP 1–7 by ACE was evident on prolonged incubation yielding to the formation of BK 1–5 (m/z 572.3) and HP 1–5 fragments (m/z 603.3), respectively (not shown). Both rHP and BK peptides are degraded in a time-dependent fashion with more than 90% being

Discussion

The comparative analysis of the blood pressure modulating effects of rHP and BK revealed that rHP exhibits hypotensive activities similar to BK, in agreement with the findings of Rioli et al. [18]; its potency, however, on a molar basis is much lower than initially reported (inasmuch when injected i.v. as i.a.), at least in the rat [18]. We extended these findings to other animal models, such as rabbits and mice. The reasons for the apparent difference of HP biological activities between the

Acknowledgements

The authors wish to thank Drs. S. Salvadori and R. Guerrini (University of Ferrara, Italy) for providing a quantity of HP. F. Gobeil is a recipient of a Junior 1 Scholarship from the Fonds de la recherche en santé du Québec (FRSQ). This study was supported in part by grants from the Canadian Institute of Health Research (CIHR; Grant MOP #66998) and the Heart and Stroke Foundation of Québec and from starting funds from the Faculty of Medicine of the Université de Sherbrooke.

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    Both authors contributed equally to this work.

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