Elsevier

Peptides

Volume 24, Issue 8, August 2003, Pages 1201-1206
Peptides

In vitro metabolism of LVV-Hemorphin-7 by renal cytosol and purified prolyl endopeptidase

https://doi.org/10.1016/j.peptides.2003.07.005Get rights and content

Abstract

The metabolism of LVVH7, an endogenous peptide obtained by cathepsin D hydrolysis of the β chain of hemoglobin, was studied, in vitro, in the presence of cytosol of rat kidney and compared with angiotensin IV. High metabolic activity was found against these two peptides (half life time <2 min) in this subcellular fraction. The main products of LVVH7 metabolism by renal cytosol are VVH7, H7 and LVVH6 suggesting both aminopeptidase and carboxypeptidase activities. The use of PEP inhibitor in kidney cytosol permitted to demonstrate the major role of prolyl endopeptidase (PEP) in LVVH7 degradation. This fact was reinforced by a kinetic study investigated with purified enzyme (Km/Vmax about 238 mM−1 s−1 and close to that observed for angiotensin related peptides).

Introduction

The hemorphins constitute a family of small peptides showing affinity for opioid receptors and exhibiting morphinomimetic properties [19], [24]. They were obtained in vitro by peptic hydrolysis of a specific zone of the β-chain of hemoglobin (residues 31–40 of bovine hemoglobin corresponding to residues 32–41 of human hemoglobin) suggesting that they could be also generated in vivo during hemoglobin proteolyis process [20]. Thus these peptides were found in large amounts in a variety of mammalian tissues (hypothalamus [4], pituitary [8]) or fluids (plasma [10], cerebrospinal [9], serum (not published), gingival crevicular [16] and bronchoalveolar lavage fluid [3]) under physiological or pathological conditions.

In addition to their opioid like activity, several other biological activities of hemorphins have been observed. Many studies seem to demonstrate that they could interact with the renin–angiotensin system (RAS) which plays a pivotal role in cardiovascular homeostasis, hydroelectrolyte balance and cell function. It was notably demonstrated that intravenous injection of Hemorphin 4 in rats leads to a significant decrease of the mean arterial blood pressure [14]. Moreover, it was also shown that hemorphins could inhibit activity of Angiotensin Converting Enzyme (ACE), an exopeptidase that converts the inactive decapeptide angiotensin I into the potent vasopressor octapeptide, angiotensin II [13], [25]. The most effective hemorphin inhibiting ACE activity is LVV-Hemorphin-7 (L-V-V-Y-P-W-T-Q-R-F). Moreover, the idea that hemorphins could interact with the RAS was reinforced by the observations that LVV-Hemorphin-7 (LVVH7) shares with Angiotensin IV (AgIV) both the same receptor (named AT4 receptor [17]), and the possibility to be inactivated by aminopeptidase N degradation [7]. Nowadays, LVVH7 like AgIV is considered as an endogenous AT4 ligand.

Whereas physiological or pathological presence and/or potential activities of hemorphins had been extensively studied, only few studies are related to their metabolism. It was suggested that cathepsin D could represent the first step of their generation with release of LVVH7 and VVH7 [5], [6]. These peptides could become secondly substrate for exoprotease, diaminopeptidase and/or aminopeptidase. However, the proteolytic pathway of hemorphins metabolism could be specific of each tissue and is still unknown [11], [22]. Because kidney represents a target for these peptides, we attempted to examine if it could also represent a degradation site of these peptides. In this context, the aim of this study was to investigate the renal cytosolic metabolism of one particular hemorphin, LVVH7 and compare it with an angiotensin peptide, AgIV. Because of presence of one prolyl residue in their structure, we were interested in the potentiality of a purified prolyl endopeptidase (PEP) to degrade these two peptides. Indeed, PEP (EC 3.4.21.26) is a serine protease which acts as a post-proline cleaving enzyme. It is widely distributed among mammalian and bacterial sources and most commonly reported to be cytosolic [1]. The presence of the enzyme in serum and seminal plasma suggests that a secreted form could exist.

The wide distribution of PEP and its activity towards many bioactive peptides [1] (angiotensin I, bradykinin, neurotensin, substance P, vasopressin) imply that the enzyme may have biological functions, notably a special function in the regulation of peptide hormone action by its role in the in vivo inactivation of oxytocin and vasopressin.

The use of a PEP inhibitor in kidney cytosol enabled us to demonstrate the major role of PEP in degradation of LVVH7. A kinetic study was also investigated with purified PEP.

Section snippets

Reagents

Hemorphins (Table 1) were synthetized by Altergen (Strasbourg, France). Bovine serum albumin (BSA), kits for marker enzymes (ALP, Sigma procedure 245; ACP, Sigma procedure 435; LDH, Sigma procedure 500), trypsin inhibitor and bestatin were from Sigma, Z-Pro-Pro-OH, and angiotensin IV from Bachem. The purified PEP (EC 3.4.21.26) was purchased by Seikagaku corporation (USA).

The liquid chromatographic system consisted of a Waters Alliance 2690 automated module equipped with a photodiode array

Renal cytosolic metabolism of LVVH7

Rat renal cytosolic metabolism of LVVH7 and AgIV were investigated in vitro. As shown in Fig. 1, AgIV and LVVH7 are rapidly degraded, with similar decay rates (half life time of 1 min 42 s for LVVH7 and 1 min 55 s for AgIV). Among the products released from LVVH7 hydrolysis by renal cytosol, four hemorphins have been identified: LVVH6, VVH7, H7, and VVH6 (Fig. 2), suggesting implication of both aminopeptidase and carboxypeptidase activities in the renal cytosolic degradation of LVVH7. The use of

Discussion

Hemorphins are bioactive peptides derived from hemoglobin hydrolysis. They could be generated in the organism during physiological [12] (catabolism of the red blood cells) or physiopathological [21] (inflammation) hemoglobin proteolysis. In this context, the way of natural generation of hemorphins in the organism had been researched. It was demonstrated that globin catabolism by macrophages could be a possible pathway for in vivo hemorphins appearance [2]. Thus, a proteolytic pathway

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