Behavioral effects evoked by the beta globin-derived nonapeptide LVV-H6

Highlights

• LVV-h6 increased the number of entries and the time spent in open arms of the maze, an indicative of anxiolysis.

• LVV-h6 provoked antidepressant effect in the forced swimming test.

• LVV-h6 increased the exploration.

• LVV-h6 did not change the cardiovascular and neuroendocrine reactivities to acute stress.

Abstract

LVV-hemorphin-6 (LVV-h6) is bioactive peptide and is a product of the degradation of hemoglobin. Since LVV-h6 effects are possibly mediated by opioid or AT4/IRAP receptors, we hypothesized that LVV-h6 would modify behavior. We evaluated whether LVV-h6 affects: i) anxiety-like behavior and locomotion; ii) depression-like behavior; iii) cardiovascular and neuroendocrine reactivity to emotional stress. Male Wistar rats ( ± 300 g) received LVV-h6 (153 nmol/kg i.p.) or vehicle (NaCl 0.9% i.p.). We used: i) open field (OF) test for locomotion; ii) elevated plus maze (EPM) for anxiety-like behavior; iii) forced swimming test (FST) for depression-like behavior and iv) air jet for cardiovascular and neuroendocrine reactivity to stress. Diazepam (2 mg/kg i.p.) and imipramine (15 mg/kg i.p.) were used as positive control for EPM and FST, respectively. To evaluate the LVV-h6 mechanisms, we used: the antagonist of oxytocin (OT) receptors (atosiban – ATS 1 and 0.1 mg/kg i.p.); the inhibitor of tyrosine hydroxylase (Alpha-methyl-p-tyrosine – AMPT 200 mg/kg i.p.) to investigate the involvement of catecholaminergic paths; and the antagonist of opioid receptors (naltrexone – NTX 0.3 mg/kg s.c.). We found that LVV-h6: i) evoked anxiolytic-like effect; ii) evoked antidepressant-like effect in the FST; and iii) did not change the locomotion, neuroendocrine and cardiovascular responses to stress. The LVV-h6 anxiolytic-like effect was not reverted by ATS and AMPT. However, the antidepressant effects were reverted only by NTX. Hence, our findings demonstrate that LVV-h6 modulates anxiety-like behavior by routes that are not oxytocinergic, catecholaminergic or opioid. The antidepressant-like effects of LVV-h6 rely on opioid pathways.

Introduction

LVV-hemorphine-6 (LVV-h6) is a nonapeptide corresponding to the 32–40 fragment of the β-globin (Leu-Val-Val-Tyr-Pro-Trp-Thr-Gln-Arg) that results from the action of chymotrypsin-like enzymes [37]. LVV-h6 was isolated and identified in the pituitary gland [37], lung and heart of healthy humans [90]. Interestingly, LVV-h6 levels are elevated in the brain of patients with Alzheimer's disease [1]. The binding of LVV-h6 to μ- and σ-opioid receptors results in significant analgesic actions [37]. It was also found that LVV-h6 is able to inhibit angiotensin converting enzyme (ACE) activity [49], which suggests a role in the cardiovascular homeostasis.

Recently, we showed that LVV-h7, a decapeptide corresponding to the fragment 32–41 of β-globin human (Leu-Val-Val-Tyr-Pro-Trp-Thr-Gln-Arg-Phe), promoted anxiolytic-like and antidepressant-like effects [27]. Although there is a noticeable similarity when comparing the primary structure between the deca- and the nonapeptide, LVV-h6 lacks a phenylalanine in the C-terminus position [38]. The decapeptide (LVV-h7) is an agonist of angiotensin IV (Ang IV) receptor (AT4), a receptor with a catalytic domain that belongs to the class of insulin-regulated aminopeptidases (IRAP), capable of degrading several molecules, including oxytocin (OT) [11,41,53,58,83]. The activation of AT4/IRAP by the agonists Ang IV and LVV-h7 could inhibit the catalytic domain and reduce the oxytocinase activity [11,6061].

AT4/IRAP is expressed in brain regions such as the prefrontal, insular and entorhinal cortices, substantia nigra, hypothalamus and amygdala [22,39], dorsal root ganglia and spinal cord, with greater ventricular expression [23]. In the human brain, the AT4/IRAP was found in Meynert basal nuclei, in the hippocampus and neocortex [21]. Therefore, it is possible that LVV-h6 and LVV-h7 play a role in the control of several physiological functions, probably reaching central AT4/IRAP and other targets to exert analgesic [38] and behavioral effects [27]. Recently, we reported that the behavioral effects evoked by LVV-h7 depend on oxytocin receptors (OTr). This result suggests that the agonism of central AT4/IRAP by LVV-h7 increases OTr activation, probably resulting from increases central OT levels [27].

Studies have revealed Ang IV as a competitive inhibitor of IRAP, since it binds to this receptor and modifies the catalytic activity [53]. A synthetic analog of LVV-h7 without the amino acid Valine at position 3 (desVal3-LVV-h7) was unable to interact with the receptor/enzyme. These findings demonstrated that: i) N-terminal portion of LVV-h7 interacts with AT4/IRAP; ii) valine is crucial for the effects of LVV-h7 [52]. A synthetic peptide with the last four amino acid residues (Thr-Gln-Arg-Phe) from the C-terminal portion of LVV-h7 did not modify the binding affinity of the decapeptide for IRAP [52]. In addition, modifications at the terminal C-8 terminus did not promote significant changes in the Ang IV affinity for AT4/IRAP [72]. Val-Tyr-Pro-Trp-Thr is the minimum sequence required to display a suitable affinity for IRAP [52], which is comprised in both LVV-h6 and LVV-h7. Since LVV-h7 evokes behavioral effects that are related to the agonism of central AT4/IRAP, as consequence, such effects are partially dependent on OTr [27], we hypothesized that LVV-h6 would evoke behavioral effects potentially mediated by opioid receptors or by AT4/IRAP. Considering that both LVV-h6 and LVV-h7 have the minimal amino acid residue sequence required for presenting high affinity to AT4/IRAP, the main aim of this study was to test whether LVV-h6 would affect: i) anxiety-like behavior and locomotion/exploration; ii) depression-like behavior; iii) cardiovascular and neuroendocrine reactivity to acute emotional stress. We also checked whether LVV-h6 effects would be similar to those reported for LVV-h7 [27], mediated by OTr activation that result from reductions in oxytocin degradation by an allosteric inhibition upon AT4/IRAP catalytic domain [83]. These possible effects would rely on: i) opioid pathway, since LVV-h6 presents affinity for the opioid receptors [3738]; ii) catecholaminergic pathway, in regards to their modulatory effects upon behavior [5].