Hemorphin and hemorphin-like peptides isolated from dog pancreas and sheep brain are able to potentiate bradykinin activity in vivo

Abstract

Hemorphins are biologically active peptides, derived from hemoglobin, which presents a number of physiological activities. Proteolytic generation of these peptides is not fully understood; however, among their roles, is to provoke reduction on blood pressure. In this work, this particular biological effect was chosen as the monitor for the selection of mammalian vasoactive peptides. By combining high-performance liquid chromatography and mass spectrometry, including ‘de novo’ sequencing, several hemorphin-like peptides were identified presenting bradykinin potentiating activity. Moreover, taking LVV-hemorphin-7 as model compound, we evaluated its biological effect on blood pressure of anaesthetized rats. By summarizing all the results, it is possible to present the hemorphins as a family of proteolytically generated peptides that are able to potentiate bradykinin activity in vivo.

Introduction

Recent advances in understanding the chemistry of life have shown that, during the course of evolution, one particular interaction between biomolecules has played a fundamental role in the physico-chemical mechanisms by which physiological processes are triggered and modulated: the peptide–macromolecule interaction [20]. Limited proteolysis of precursor proteins derived either from endogenous or exogenous sources is the source of these essential bioactive peptides. The bioactive peptide hormones may be generated intra- or extra-cellularly, and may act as well in both the compartments. Hemorphins are a typical case: Rioli et al. [37] have identified these peptides intracellularly in the brain and this work presents an effect that is believed to be mainly extra-cellular.

Hemorphins are endogenous peptides belonging to the family of “non-classical” or “atypical” opioid peptides that can be enzymatically released in vitro from hemoglobin by macrophages [11], by endogenous lysosomal proteases [12], by pepsin [36], [9], by pancreatic elastase [23] and by cathepsin D [16], [13]. The hemorphin family member peptides vary in size from 4 to 10 amino acids and their main motif is comprised in the 32–41 segment of human hemoglobin β-chain. Moreover, Piot et al. [36] isolated a fragment of six amino acids from α-chain of bovine hemoglobin, which was able to potentiate bradykinin in vitro. However, the in vivo mechanism for the generation and release of these peptides has not been clarified in its full details (see Ref. [31] for a review on the subject). These peptides have been isolated from a number of tissues and fluids, including hypothalamus, pituitary, plasma, serum and cerebrospinal fluid, but the composition and content for each tissue or fluid seem to be characteristic [31], [22], [18].

On the other hand, hemorphins can be degraded (or processed) by the cytosol of renal tissue [15], prolyl oligopeptidase [15], cathepsin B [13], endopeptidase 24.15 and neurolysin [37] and dipeptidyl peptidase IV [10]. Moreover, hemorphins are inhibitors of insulin-regulated amino peptidase, IRAP, [26], angiotensin-converting enzyme, ACE, [24], [14] and dipeptidyl peptidase III [7].

Hemorphins present a number of biological activities, including effects on spatial learning [25], analgesia [27], transient hypotension [2], constriction of coronary vessels, platelet aggregation, stimulation of calmodulin-depend enzymes [28], pressor effect and tachycardia [3], the interaction with opioid receptors [2], [3] and inflammation [38]. Moreover, a recent study [5] was able to demonstrate that LVV-hemorphin-7 can directly reduce blood pressure in spontaneous hypertensive rats.

The kallikrein–kinin system, having bradykinin lying at its center, represents a metabolic cascade that, when activated, triggers the release of vasoactive kinins. This complex multi-protein system includes the serine proteases tissue and plasma kallikreins, which liberate kinins from high- and low-molecular weight kininogen (HK and LK). Kinins exert their pharmacological activities by binding specific receptors, before being metabolized by various peptidases. The autocrine or paracrine activity of kinins is regulated by several metallopeptidases, the relative importance of which varies from one biological medium to the other. Finally, the regulation of this system by serpins adds to the complexity of the system, as well as its multiple relationships with other important metabolic pathways such as the renin–angiotensin, coagulation or complement pathways (for a review on the subject, see the work of [29]). Nevertheless, the bradykinin potentiating peptides (BPPs), extremely active and widely present on snake venoms, have not, so far, been found as an endogenous component of this system [19].

Moreover, external ‘players’ to the system may also assume important roles on the local regulation of the kallikrein–kinin system, as their generation may not be subjected to a fine control as the whole system is. Rather, their generation may be related to a traumatic event, such as tissue damage, as for instance, it is the case of the generation of the antimicrobial peptide parasin I from histone H2 by cathepsin D in the wounded skin of catfish [8]. These peptides, quickly and locally generated in situ may function as the external ‘players’ that help the unbalanced system to recover its homeostasis.

In the present study, we report the isolation of hemorphins from the low Mr fraction of dog pancreas and sheep brain homogenates. The peptides present in the positive fractions for the biological assays, eliciting smooth muscle contraction by potentiation of bradykinin activity, were ‘de novo’ sequenced and have demonstrated to be hemorphin and hemorphin-like molecules, as identified by their typical amino acid sequence.