Signaling molecules targeting cannabinoid receptors: Hemopressin and related peptides

Journal Pre-proof Signaling molecules targeting Hemopressin and related peptides

cannabinoid

receptors:

Fengmei Wei, Long Zhao, Yuhong Jing PII:

S0143-4179(19)30079-4

DOI:

https://doi.org/10.1016/j.npep.2019.101998

Reference:

YNPEP 101998

To appear in:

Neuropeptides

Received date:

20 May 2019

Revised date:

21 November 2019

Accepted date:

21 November 2019

Please cite this article as: F. Wei, L. Zhao and Y. Jing, Signaling molecules targeting cannabinoid receptors: Hemopressin and related peptides, Neuropeptides(2018), https://doi.org/10.1016/j.npep.2019.101998

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© 2018 Published by Elsevier.

Journal Pre-proof Signaling molecules targeting cannabinoid receptors: hemopressin and related peptides Fengmei Wei a, Long Zhao b, Yuhong Jing c* a Department

of Physiology and Psychology, School of Basic Medical Sciences, Lanzhou University, Lanzhou, Gansu Province 730000, PR China

b

Department of Orthopaedics, Lanzhou University First Affiliated Hospital, Lanzhou, Gansu Province 730000, PR China

c

Institute of Anatomy and Histology & Embryology, Neuroscience, School of Basic Medical Sciences, Lanzhou University, Lanzhou, Gansu, 730000, PR China; Key Laboratory of Preclinical Study for New Drugs of Gansu Province, Lanzhou University, Lanzhou, Gansu, 730000, PR China * Corresponding

author at: Institute of Anatomy and Histology & Embryology, Neuroscience, School of Basic

Medical Sciences, Lanzhou University, Lanzhou, Gansu, 730000, PR China; Key Laboratory of Preclinical Study

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E-mail address: [email protected] (Yuhong Jing)

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for New Drugs of Gansu Province, Lanzhou University, Lanzhou, Gansu, 730000, PR China

Journal Pre-proof 2-AG, 2-arachidonoyl glycerol; Aβ1–42, amyloid-β (1–42); ACE, angiotensin-converting enzyme; BBB, blood–brain barrier; BK, bradykinin; CB, cannabinoid; CBRs, cannabinoid receptors; Cg, carrageenan; CNS, central nervous system, CPP, conditioned place preference; DAMGO, [D-Ala2, NMe-Phe4, Gly-ol5]-enkephalin; ep24.15, endopeptidase EC 3.4.24.15; ep24.16, endopeptidase EC 3.4.24.16; ERK, extracellular signal-regulated kinase; GI, gastrointestinal; Hb, hemoglobin; Hp, hemopressin; i.a., intra-arterial; i.v., intravenous injection; i.t., intrathecal injection; i.p., intraperitoneal injection; i.c.v., intracerebroventricular injection; i.pl., intraplanar injection; NAM, negative allosteric modulator; NE, norepinephrine; p.o., per os; PAM, positive allosteric modulator; PBS, phosphate-

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buffered saline; NaP, sodium phosphate buffer; TRPV1, transient receptor potential vanilloid type 1

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Journal Pre-proof 1 Introduction 1.1 Cannabinoid receptors (CBRs) CBRs are part of the endocannabinoid system; this system is involved in numerous physiological processes such as nociception, inflammation, appetite, stress, and emotion regulation 1–3. Many studies have linked the endocannabinoid system to neuroinflammatory and neurodegenerative disorders such as Parkinson’s disease, Huntington’s chorea, Alzheimer’s disease 4,5, multiple sclerosis 6, and other diseases 7. Thus, the modulation of CBRs by selective drugs poses intriguing opportunities for managing these diseases. There are two major subtypes of CBRs, CB1 receptor (CB1R) and CB2 receptor (CB2R), which are members of the G protein-coupled receptor family. At the gene level, human CB1R is located on chromosome 6q14–15; human CB2R is located on chromosome 1p36 8. Initially, CB1R was known to be mainly expressed in the central nervous system (CNS), but later it was identified in almost all peripheral tissues and cell types 7,9. CB2R is mainly expressed in the immune system and in hematopoietic cells, but it is also detected in some other peripheral tissues At the protein level, CB1R and CB2R share 44% amino acid homology 8,10. The crystal structures of CB1R 11,12

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9.

(PDB ID: 5TGZ) and CB2R 13 (PDB ID: 5ZTY) were determined using AM6538 and AM10257 as CB1R and CB2R stabilizing antagonists, respectively. The ligand-binding pockets of CB1R and CB2R show strong similarities; therefore, it is difficult to design subtype-specific ligands 14.

It is well known that the primary psychoactive constituent of marijuana, i.e., Δ9 tetrahydrocannabinol, and the

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endogenous cannabinoids (endocannabinoids) anandamide and 2-arachidonoyl glycerol (2-AG) 8 are CBR agonists. Agonist–receptor interactions result in the activation of G proteins, particularly of the Gi/o family 15

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(Figure 1). Activation of Gi/o will lead to inhibit cAMP production, decrease Ca2+ conductance, and increase K+ conductance and mitogen-activated protein kinase activity 16. In addition to Gi/o-mediated signaling, CB1R and CB2R could be phosphorylated by G protein-coupled receptor kinases and subsequently associate with β-arrestin

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1 or 2 8, which can serve as a scaffold for their interaction with proteins that divert the signaling along β-arrestinmediated pathways. Then the interaction between Gβγ and β-arrestin participated in extracellular signal-regulated

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kinase (ERK) 1 and 2 stimulated by CB1R and CB2R 8.

Figure 1 Signaling pathways of CBR activation.

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Journal Pre-proof 1.2 Hemopressin (Hp) and related peptides Rat Hp [PVNFKFLSH, (r)Hp], a fragment of the Hb-α1 chain (95–103 amino acids), is an intracellular peptide isolated from rat brain and spleen homogenized in boiling acid buffer 17. It is interesting that using an acidfree alternative method only the N-terminally extended peptides of Hp RVD-Hpα (RVDPVNFKLLSH) and VDHpα (VDPVNFKLLSH) as well as VD-Hpβ (which is derived from the hemoglobin β chain) were identified in the extracted brain peptides 18. The similar result was also present in an immunoaffinity mass spectrometry-based study in which more N-terminally extended peptides named pepcans (peptide endocannabinoids) were identified in the brain and plasma and Hp remained unidentified 19. So, Hp was postulated to be generated from the cleavage of longer peptides under acidic conditions 10,18,20. RVD-Hpα, the N-terminally extended peptides of Hp, which was found in the studies 18 19 mentioned above, was considered the endogenous peptide 18,19. It is appealing to clarify the functions of the endogenous peptide. Due to its distribution in both brain and peripheral blood, RVD-Hpα was postulated to be a neuropeptide functioning as a cell–cell signaling molecule 21. It was supported that RVD-Hpα

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could be secreted from the cultured mouse brain slices 22. It was also supported that the source of RVD-Hpα in the peripheral might be chromaffin cells in the adrenal glands 23. In addition, the pharmacological activities of RVDHpα were identified as a negative allosteric modulator (NAM) of CB1R 19 in the brain or a positive allosteric modulator (PAM) of CB2R 24 in the peripheral.

In this review, many issues surrounding Hp, RVD-Hpα, and their related peptides, such as their discovery,

activities, are discussed.

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2 Discovery of Hp and related peptides

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structure, metabolism, brain exposure, self-assembly characteristics, and pharmacological characterization and

2.1 Hp

Intracellular peptides are produced by proteasomes following the degradation of nuclear, cytosolic, and

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mitochondrial proteins. They can be further processed by additional peptidases, generating a large pool of peptides within cells 25. Endopeptidase EC 3.4.24.15 (ep24.15; also referred to as thimet oligopeptidase) and endopeptidase

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EC 3.4.24.16 (ep24.16; also referred to as neurolysin), widely expressed in the mammalian brain, are closely related metalloendopeptidases implicated in the metabolism of several neuropeptides. Using the inactive forms of ep24.15 and ep24.16 in a substrate capture assay, a total of 15 ep24.15 and ep24.16 binding peptides were isolated from peptide extracts of rat brain and spleen homogenates, which were boiled in acid buffer for 10 min. Hp was

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the peptide that was effectively hydrolyzed by angiotensin-converting enzyme (ACE). Moreover, Hp induced hypotension when administered through the left femoral vein in anesthetized rats. Accordingly, Hp received its name 17.

2.2 RVD-Hpα and VD-Hpα RVD-Hpα and VD-Hpα were discovered by Gomes et al. while clarifying the source of Hp 18. Because the Asp–Pro bond is labile under acidic conditions 18, it was uncertain if Hp was an endogenous peptide because it was identified under acidic conditions. Therefore, in their procedure, an alternate method that did not involve acidic conditions was used to extract mouse brain peptides 18. Subsequently, no Hp was found, but the N-terminally extended peptides of Hp RVD-Hpα, VD-Hpα, and VD-Hpβ were identified. Therefore, RVD-Hpα and VD-Hpα were considered endogenous peptides 18,26. Later, using LC/MS/MS with optimized protein precipitation and solidphase extraction methods, it was also shown that not Hp but RVD-Hpα (pepcan-12) and pepcan-23 (possible precursor of pepcan-12) were quantified at physiologically relevant levels in the tissues 24. 2.3 Pepcans Pepcans are a series of N-terminally extended peptides of (m)RVD-Hpα. They were discovered during the study on CB1R binding for the endogenous peptide (m)RVD-Hpα. In the procedure, monoclonal antibodies at the 4

Journal Pre-proof C-terminal end of (m)RVD-Hpα were prepared; then, in immunoaffinity mass spectrometry-based experiment, pepcan-12 to pepcan-23 were identified in mouse brain as well as mouse and human plasma samples 19. In the brain, because pepcans are located in the noradrenergic neurons of the locus coeruleus 23, promising that these peptides play critical roles in signal transduction in the brain. The sample preparations used in discovering Hp and related peptides are shown in Table 1. Hp was found in the brain and spleen samples 17, but later studies did not contain spleen samples. Thus, it is possible that Hp can be obtained from the spleen, which plays important roles in RBC clearance. According to the peptide structure, Hp is stable at acidic pH, whereas RVD-Hpα is stable at pH 3.0 or pH 7.4 27; therefore, it is possible that the acid extraction method is suitable to identify Hp. Moreover, Hp can be easily hydrolyzed by enzymes such as ACE, which is distributed throughout the body; therefore, it is possible that Hp is hydrolyzed immediately after its production. Moreover, the finding that only (m)RVD-Hpα and (m)VD-Hpα were identified in the buffer at pH 9.5 may be because other pepcans were not stable in the same buffer. Thus, owing to the differences in the sample

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sources and the buffer used to prepare the samples, particularly the pH of the buffer, it is difficult to conclude if Hp is an artifact during hot acid extraction or exits under physiological conditions. However, because RVD-Hpα is stable at any pH, it is possible that RVD-Hpα is the most common form of Hp-related peptide in the body. Table 1 Sample preparation in discovering Hp and related peptides Source a

Tissue b

Buffer

Hp

R

B or S

25 mM Tris-HCl, 125 mM NaCl, 0.1%

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Peptide

pH

Reference

7.5

17

9.5

18

7.2

19

7.2

19

VD-Hpα

M

B

M and R

B

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bovine serum albumin 10 mM HCl, 0.2 M phosphate buffer

Pepcans

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RVD-Hpα

PBS c, 10mM HCl, 100 mM acetic acid,

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acetonitrile (66% or 50%)

H and M

P

rat; M, mouse; H, human

b B,

brain; S, spleen; P, plasma

c

albumin, acetonitrile (66% or 50%)

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a R,

PBS, 0.05% Tween, 0.5% bovine serum

PBS, phosphate-buffered saline

3 Sequence and structure of Hp and its peptides 3.1 Hp The chemical formula and molar mass of Hp are C53H77N13O12 and 1088 Da, respectively. The sequences of Hp derived from different species differ in the amino acid present at the 100th position of the Hb-α1 chain or 6th position of Hp, i.e., Phe in rats [(r)Hp, PVNFKFLSH] and Leu in mice [(m)Hp, PVNFKLLSH], humans, pigs, and cattle 25,28. The sequences of (m)Hp and its related peptide are shown in Figure 2. Seven amino acids at the Nterminus of Hp are highly conserved among turtles, crocodiles, birds, marsupials, rodents, and mammals 29. These amino acids are considered the primary coordination sites for (r)Hp. Pro1 and Val2 at the N-terminus are required by (r)Hp to antagonize CB1R, with the C-terminus being the anchor site. Leu7–His9 found at the C-terminus does not appear essential for the biological activity of CB1R antagonism, but it can serve as an anchor site for metal ions at acidic pH 30,31. Lys6 and His9 in Hp were found to be Lys99 and His103 in α-Hb. If Lys99, His103, and Tyr117 in α5

Journal Pre-proof Hb are mutated to Ala, the interaction of α-Hb with α-Hb stabilizing protein is abrogated 32. Additionally, the Cterminus of Hp can moderately enhance the affinity for μ receptors because the displacement by Hp(1–7) is lower than that by Hp in a competitive receptor binding assay of [3H]DAMGO ([D-Ala2, NMe-Phe4, Gly-ol5]enkephalin) 33. Moreover, unlike the CB1R antagonist SR141716 (rimonabant), (r)Hp cannot penetrate into the ligand-binding pocket and aromatic ring region of CB1R 30; it can only bind to the external binding site of CB1R because of the limitation of the benzene ring 30. Recently, docking studies have showed that the nuclear magnetic resonance conformation of Hp matches the binding pocket of taranabant (a CB1R inverse agonist) in CB1R, and all residues essential for taranabant binding are in proximity to the side chains of Hp 27.

Hp (1–3)

PVN

Hp (1–4)

PVNF

Hp (1–5)

PVNFK

NFKL a Hp (1–6) Hp (1–7)

PVNFKL

Hp (1–8)

PVNFKLLS

NFKL

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PVNFKLL

Pepcan-9 or Hp

PVNFKLLSH

Pepcan-11 or VD-Hpα

VDPVNFKLLSH

Pepcan-12 or RVD-Hpα

RVDPVNFKLLSH

Pepcan-13 b

LRVDPVNFKLLSH

Pepcan-14

KLRVDPVNFKLLSH

Pepcan-15

HKLRVDPVNFKLLSH

AHKLRVDPVNFKLLSH

Pepcan-17

HAHKLRVDPVNFKLLSH

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Pepcan-16

Pepcan-18

LHAHKLRVDPVNFKLLSH

Pepcan-19

DLHAHKLRVDPVNFKLLSH

Pepcan-20

SDLHAHKLRVDPVNFKLLSH

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Pepcan-21

LSDLHAHKLRVDPVNFKLLSH ALSDLHAHKLRVDPVNFKLLSH

Pepcan-23

SALSDLHAHKLRVDPVNFKLLSH

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Pepcan-22

a NFKL,

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Figure 2 The sequence of mouse Hp and related peptide. Asn3–Leu6 fragment of mouse Hp (Orange); b has not been detected in the existing studies.

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3.2 Hp terminally truncated derivatives The sequence of mouse Hp terminally truncated derivatives is shown in Figure 2. It appears that the full sequence of Hp is unnecessary for its bioactivity. (r)Hp(1–6) (PVNFKF) and (r)Hp(1–7) (PVNFKFL) were as effective as (r)Hp for carrageenan (Cg)-induced hyperalgesia, but Hp(1–5) (PVNFK) and Hp(1–4) (PVNF) had no such activity 34,35. Therefore, (r)Hp(1–6) was initially regarded as the shortest fragment that retains full activity 34,35.

The structure of (r)Hp(1–6), similar to (r)Hp, assumes random coil conformation with minimal amounts of

turn and strand structures in a water solution 30. In a micelle solution, both (r)Hp(1–6) and (r)Hp assume ordered secondary structures. Remarkably, regular β-turn structures are present in the Asn3–His9 segment of (r)Hp and the Asn3–Phe6 segment of (r)Hp(1–6), suggesting that Asn3–Phe6 contain the essential chemical moiety that interacts with CB1R 30. The Asn3–Phe6 fragment of (r)Hp (NFKF) has a better Goldscore for binding to CB1R than that for binding to AM6538 36. NFKF is expected to be a potent oral medicine to control the symptoms of pilocarpineinduced epileptic seizures 36. NFKL has properties similar to those of NFKF 36, but shorter fragments, such as NFK, FKL, NF, FK, KF, or KL, have no oral pharmacological activity in preventing or altering seizures 36. 3.3 Hp N-terminally extended peptides The sequence of mouse Hp N-terminally extended peptides is shown in Figure 2. Although VD-Hpα and RVD-Hpα are 2–3 amino acids longer than Hp, some of their activities appear opposite to each other. First, since 6

Journal Pre-proof 2007, Hp had been regarded as an antagonist or inverse agonist of CB1R 29, but in 2009, RVD-Hpα and VD-Hpα were considered CB1R agonists 18 and subsequently a NAM 19 of CB1R and PAM of CB2R, respectively 24. Second, the stability of the two peptides was distinguished under different pH values. The His sidechain in Hp was protonated, which stabilized the helical structure at the C-terminus (C-capping) at pH 3.0, but this did not occur at pH 7.4 because the His sidechain was likely to be uncharged 27. In contrast, RVD-Hpα is more stable than Hp at pH 7.4, because it possesses the stabilizing influence of the Asp negative charge at the N-terminus (N-capping). Thus, in nuclear magnetic resonance studies, Hp exhibits a helical structure that is favored at an acidic pH, whereas RVD-Hpα exhibits a helical structure that is favored at a neutral pH 27. This may be the reason that Hp is derived following hot acid extraction, 17 but it disappears when an acid-free extraction method is used 18,26. Third, the opposite activities of Hp and RVD-Hpα were also found in their self-assembly characteristics 20, which is mentioned in issue 7. Longer N-terminally extension peptides, such as other pepcans, also exhibit different activities compared with RVD-Hpα (pepcan-12). Pepcan-13 has not been detected in the existing studies, and 24.

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pepcan-14, -15, and -17 have not been found to be as effective as RVD-Hpα in CB2R binding interaction studies Thus, the added amino acids at the N-terminus of Hp could help determine the stability and pharmacological

characterizations of Hp N-terminally extended peptides. 4 Metabolism of Hp and related peptides

Under normal conditions, aging and defective red blood cells are removed from the circulation by the

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phagocytic activity of macrophages in the spleen or liver or are hemolyzed within the circulation. Heme and globin are produced during the rupture of RBCs. Heme degradation leads to carbon monoxide release, iron recycling, and bilirubin production. Globin, including α-globin and β-globin, undergoes intense proteolytic degradation and

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produces peptides 25. Peptides sized 3–22 amino acid residues are generated from globin during the ubiquitindependent (3–14 amino acid residues) and ubiquitin-independent (3–22 amino acid residues) intracellular proteolysis of 20S proteasomes 37,38. In addition to 20S proteasomes, other intracellular proteases, such as calpains,

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caspases, and cathepsins, participate in globin proteolysis 39. Subsequently, enzymes such as ep24.15 and ep24.16 with substrates of the optimum size range (i.e., 5–17 amino acids) hydrolyze these peptides into smaller peptides some of which are bioactive.

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17,

Hp serves as a substrate for ep24.15, ep24.16, and ACE in brain and spleen tissues 17. These three enzymes belong to thermolysin-like metalloendopeptidases and have similar structural aspects 40. Among them, ep24.15 is

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widely distributed in mammalian tissues and is highly expressed in the brain, pituitary, and testis, which are tissues rich in neuropeptide content 41. Ep24.16 is distributed ubiquitously, with the lowest peptidase activity in the heart and the highest activity in the liver and kidneys in mice 42. Although primarily cytosolic enzymes, ep24.15 and ep24.16 were reported to be secreted by cells and to exhibit plasma membrane-associated forms, which allow them to degrade extracellular peptides 43,44. Within the vasculature, ep24.15 and ep24.16 share many natural substrates such as bradykinin (BK), angiotensin I, opioids, and gonadotrophin-releasing hormone 17. Hp is a better substrate for ep24.15 and ep24.16 than BK 17, and both enzymes show high affinity for Hp. ACE localizes on the surface of various cells and is distributed throughout the body. It is well known that ACE converts angiotensin I to angiotensin II, and metabolizes kinins as well as many other biologically active peptides, including substance P, chemotactic peptides, and opioid peptides 45. ACE hydrolyzes Hp more efficiently than it hydrolyzes angiotensin I or BK 17. Thus, once these enzymes are active, Hp can be easily hydrolyzed in tissues or within their vasculature, thereby sharing a very short half-life with BK 46 if it is endogenous. Similarly, RVD-Hpα can be hydrolyzed, but owing to the stability of the structure of RVD-Hpα, RVD-Hpα is more stable than Hp, which makes it detectable in tissues 24. In mouse, RVD-Hpα was detected at the levels of 50–130 pmol/g in the adrenal gland, liver, and spleen; 20–60 pmol/g in the kidneys and lungs; and 2–3 pmol/g in the brain 24.

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Journal Pre-proof Similar to the metabolic cascade of angiotensin (Figure 3A), we hypothesize that RVD-Hpα is hydrolyzed by various enzymes, thereby generating active peptides. The conceived cascade of RVD-Hpα is shown in Figure 3B. Although several pepcans have been discovered, only pepcan-23 and pepcan-12 occur at quantifiable and physiologically relevant levels in tissues as measured by LC–MS/MS with optimized protein precipitation and solid-phase extraction 24. Pepcan-23 is detected abundantly in the brain but absent in adrenals which contained high amounts of pepcan-12, in addition to not directly modulating CBRs, thereby being postulated to be a precursor of pepcan-12 24. Pepcan-23 might be the pepcan species detected in the abundant locus coeruleus projecting fibers in the brain 23,24. Pepcan-12 or RVD-Hpα and SALSDLHAHKL are postulated to be the products of pepcan-23. If VD-Hpα and Hp exist in tissues, they may be the products of RVD-Hpα or pepcan-23. Moreover, in one study, RVD-Hpα and RVDPVNFKL were found in mouse brain slices and culture media, whereas RVDPVNFKLL and RVDPVNF were only found in the culture media 22. This indicates that RVD-Hpα is degraded from the C-terminus, and that the products of RVD-Hpα play some roles in cell–cell signaling.

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Comparing with those in the brains of C57BL/6 wild-type mice, the levels of RVD-Hpα, RVDPVNFKLL, and RVDPVNF in the brains of ep24.16-knockout mice increased 47, suggesting that ep24.16 participates in the degradation of the amino acids present at the C-terminus of these peptides in the brain. Thus, it is reasonable to believe that neuropeptidase-mediated hydrolysis of RVD-Hpα, which is similar to angiotensin, is harmoniously modulated in tissues and vessels. However, many questions regarding the source and metabolites of Hp and related peptides are yet to be answered. These questions include whether 20S proteasomes proteolyze Hp and related

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peptides and if so, then how does this occur; whether Hp is an endogenous peptide; whether there is a mechanism to protect Hp and related peptides from hydrolysis; what roles do the adrenal gland, liver, and kidneys play in the

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metabolism of Hp and related peptides; and whether Hp and related peptides in the CNS are generated in the CNS

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or in peripheral tissues.

Figure 3 Metabolic cascades of angiotensin (blue) and RVD-Hpα (orange) Moreover, because RVD-Hpα can be secreted from cultured mouse brain slices 22, in addition to existing in either the brain or peripheral blood 21, it is possible that it functions as a nonclassical neuropeptide 39. Nonclassical neuropeptides are determined based on the criteria that a peptide is synthesized in the brain, is secreted in physiologically relevant levels, has regulated synthesis or secretion, and can influence the function of another cell 39.

However, in a later study, ~0.6 ng/mg RVD-Hpα was still detected in the perfused adrenal glands but not in the 8

Journal Pre-proof perfused brain and spinal cord, supporting that RVD-Hpα was produced and released by the chromaffin cells present in the adrenal glands 23. Because Hb is found in brain cells 48,49, and intact RVD-Hpα cannot cross the blood–brain barrier (BBB) like Hp, we suppose that RVD-Hpα is produced by the neurons containing Hb in the brain and by chromaffin cells in the peripheral tissues. In the brain, using monoclonal antibodies against FKFLSH, which is the C-terminal part of Hp and RVD-Hpα, pepcans have been found to be distributed in noradrenergic LC as well as A1, A5, and A7 neurons and their axons, with an overlap of tyrosine hydroxylase-positive staining in LC cell bodies and the co-localization with galanin 23. Interestingly, in other studies, RVD-Hpα was shown to inhibit norepinephrine (NE) expression 50,51. Thus, it is possible that RVD-Hpα functions as a neuropeptide to inhibit NE. 5 Exposure amounts of Hp and related peptides to the brain The amount of brain exposure is critical for the therapeutic applications of Hp and related peptides. If Hp and related peptides are applied to the CNS, it is essential to optimize brain exposure to improve efficacy. If they are

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used in peripheral tissues, it is important to minimize brain exposure to reduce side effects 52, such as unnecessary impacts on the central reward route. The central focus is the ability of Hp and related peptides to pass through the BBB. The BBB acts as an anatomical gateway between the CNS and peripheral circulation that strictly regulates the entry of specific molecules from the circulation or their clearance 53. Most small-molecule drugs cannot generally cross the BBB; nevertheless, they can cross it in pharmacologically significant amounts only if (1) the

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molecular mass of the drug is <400−500 Da, and (2) the drug forms <8–10 hydrogen bonds with solvent water 54. Owing to its molecular mass (1088 Da), it is difficult for Hp and related peptides to cross the BBB. In a study, after the intraperitoneal (i.p.) administration of Hp, mass spectrometry did not show intact Hp in brain peptide

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extracts, suggesting that intact Hp cannot cross the BBB 55. However, this crossing is possible for Hp terminally truncated derivatives owing to their small molecular mass. Following systemic administration, Hp can activate feeding-related circuits in the mediobasal hypothalamus and nociceptive regions in the periaqueductal gray and

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dorsal raphe nucleus 56. If the intact Hp does not cross the BBB at concentrations of >10 nM, it is possible that the effects of shorter bioactive peptides derived from Hp are the reason for c-Fos activation observed in these specific

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brain areas following the systemic administration of Hp 56. 6 Cell-impermeant CB1R antagonist

CB1R is localized in intracellular vesicles and on the plasma membrane 57. It is well known that Hp can bind

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to CB1R on the cell plasma membrane, thereby Hp is found to inhibit neurite outgrowth in mouse neuroblastoma Neuro-2A cells 29 as well as promote differentiation and maturation of mouse neural stem cells 58. But it is difficult to know whether Hp binds to CB1R in intracellular vesicles. In a study on Hp as an inverse agonist of CB1R, Hp was not found to be as effective as the lipophilic CB1R antagonist SR141716 (able to cross membranes) at the same dose required to inhibit neurite outgrowth in Neuro-2A cells 29, but the plasma membrane impermeability of Hp was not mentioned in the study. Only in a study on intracellular CB1R, it was reported that Hp could not cross the plasma membrane 57 because Hp had a low inhibitory effect on WIN55212-mediated ERK phosphorylation compared with SR141716. Eventually, Hp was confirmed as a cell-impermeant CB1R antagonist in a study of mitochondrial CB1R by measuring the intracellular fluorescence of cells treated with a fluorescent derivative of Hp and by the spectrometric analysis of extracts from hippocampal slices incubated with Hp and the lipophilic CB1R antagonist AM251 59. Subsequently, in three studies, Hp was used as a cell-impermeant CB1R antagonist. One study found that Hp was not as effective as AM251 in blocking mitochondrial CB1R regulation induced by the selective cell-permeable CB1R agonist arachidonyl-2-chloroethylamide 60. Other two studies revealed that Hp reduced thyrotropin-releasing hormone-induced current and changed the low-threshold spike-induced slow afterdepolarization area in midline paraventricular nucleus neurons only when Hp was intracellularly applied via dialysis from a patch pipette, but not during bath application in an electrophysiological assay 61,62. Accordingly, 9

Journal Pre-proof Hp is a promising tool to study CB1R. However, to use Hp for this purpose, there is still one consideration. It appears that the short-term bath application of Hp cannot lead to the binding of Hp to CB1R, which is mainly located on the plasma membrane, because little effect has been observed for bath-applied Hp 61. 7 Self-assembly of Hp and related peptides The self-assembly of Hp may be one reason for the inexplicable activities of Hp observed in different experiments. In 2010, Gomes et al. compared the amounts of Hp and angiotensin II in a 2-kDa dialysis cassette 26; Hp was found to dimerize or oligomerize at high concentrations. The complex aggregated by Hp remained in the dialysis cassette after 24 h of dialysis against phosphate-buffered saline (PBS) 26. Subsequently, a further study revealed that while Hp self-assembled into fibrils under the condition of 1 mM Hp, 25 mM phosphate, 50 mM NaCl, and pH 7.4, RVD-Hpα did not at the same concentration 20,63. Later, four conserved changes in the peptide sequence (Val2→DVal2, Asn3→Gln3, Leu7→Npg7, and C-OH→C-NH2) were identified that could help minimize Hp aggregation 64. Remarkably, no aggregation or precipitation was found for Hp and RVD-Hpα at pH 3.0 and pH

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7.4 in a 50/50 v/v hexafluoroisopropanol/sodium phosphate buffer system 27. Because RVD-Hpα is more stable than Hp at pH 7.4 27, at pH 7.4, Hp is more likely to undergo self-assembly than RVD-Hpα 26. Coincidently, a previous study used PBS at pH 7.4 63. Apart from the influence of pH, the addition of 25% 2,2,2-trifluorethanol should be considered 63, which can promote secondary structure and possibly lead to the observation of a β-like structure 27. Moreover, the differences in buffers and final concentrations should not be neglected (Table 2). In a

e-

study using ultrafiltration tubes with a molecular weight cutoff of 2000 or 3000, the difference in the recovery of RVD-Hpα (pepcan-12) and pepcan-20 at low or high concentrations suggested that pepcans at a high concentration

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of 1 µM form aggregates of lower molecular weight in dimers or trimers 19. Table 2. Sample preparation in self-assembly studies on Hp and related peptides Peptide

Concentration

Buffer

pH

Reference

?

26

25 mM phosphate, 50 mM NaCl

7.4

63

RPMI 1640 medium, 10% FCS,

?

19

91.89 or 459.45

(r)Hp

1000

(m)RVD-

0.01, 1

Hpα

(m)Hp

penicillin, streptomycin, amphotericin

Jo

Pepcan-20

PBS

ur n

(r)Hp

al

(μM)

B

20

20 mM sodium phosphate buffer (NaP)

3.0, 7.4

27

500

20 mM NaP or in 50% v/v 156

3.0, 7.4

27

(m)RVDHpα

hexafluoroisopropanol/NaP, 5% D2O ?, unknown 8 Pharmacological characterization of Hp and RVD-Hpα 8.1. Hp is a CB1R inverse agonist Hemoglobin-derived peptides, such as LVV-hemorphins and VV-hemorphins, can affect blood pressure in addition to eliciting opioid receptor-mediated antinociceptive activity 65. Similarly, Hp has an antihyperalgesic effect in addition to hypotensive effect. However, this effect cannot be blocked by naloxone, suggesting that its 10

Journal Pre-proof mechanism is different from that of hemorphins 34. Because CB1R, primarily expressed in the CNS, is associated with pain, Heimann et al. 29 screened 17 peptides captured by ep24.15 as endogenous ligands modulating CB1R activity. In this study, Hp selectively bound to CB1R but not to CB2R, μ and δ opioid receptors, and α 2A and β2 adrenaline receptors. Compared to the CB1R antagonist SR141716, Hp caused similar attenuation of the agonistinduced increase in antibody recognition. In addition, Hp could reduce Cg-induced hyperalgesia such as that induced by the CB1R antagonist AM251. Taken together, Hp is possibly a CB1R antagonist or an inverse agonist. Subsequently, a versatile peptide research tool involving the radiolabeling of Hp(1–7) was created for the direct study of CB1R–peptide ligand interactions in 2016 66. In the study, [3H]Hp(1–7) was specifically bound to a membrane receptor but seemingly not to CB1R. A further study revealed that Hp(1–7) and Hp slightly activated G proteins and functioned as very weak agonists. Thus, Hp and Hp(1–7) may be the indirect regulators of CB1R, instead of an endogenous ligand 66. Remarkably, while studying intracellular CB1R, it was found 57 and confirmed 59

that Hp could not cross the plasma membrane. Thus, Hp was suggested to be an indirect cell-impermeant CB1R

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antagonist. 8.2 RVD-Hpα acts in a manner consistent with an allosteric modulator of CBRs In contrast to Hp, its extended peptides RVD-Hpα and VD-Hpα were initially identified to be CB1R agonists. However, the signaling pathways activated by these extended peptides differ from those activated by the endogenous cannabinoid 2-AG and the classical CB1R agonist Hu-210 18. Later, RVD-Hpα was reported as a

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potential NAM in 2012 when pepcans were discovered 19. Allosteric modulators bind to a site topographically different from the orthosteric binding site. This can increase (PAM) or decrease (NAM) the affinity and/or efficacy of an orthosteric agonist by changing protein conformation 67. It is confirmed that Hp doesn't function as a NAM

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of CB1R like RVD-Hpα in a study using electrophysiology 68. In the view of the conflict with previous descriptions, they postulated that Hp cannot cross the lipid bilayer and, therefore, was retained in the recording pipette 68. Notably, RVD-Hpα was soon identified as a PAM of CB2R in peripheral tissues 24. It was shown that

al

RVD-Hpα could induce 20%–40% increase in binding of the high-affinity ortholigands [3H]CP55940 and [3H]WIN55212-2 to CB2R; pepcan-14, pepcan-15, and pepcan-17 had weak effects; pepcan-20 and pepcan-23 had

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no effect 24. Next, it was demonstrated that RVD-Hpα exhibited the effect of CB2R-mediated G-protein recruitment and cAMP inhibition (Gα i) without affecting β-arrestin-2 recruitment and receptor internalization, whereas other pepcans did not significantly affect agonist-induced cAMP inhibition 24. It is promising that RVD-

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Hpα plays an important role in modulating immunity and inflammation which is mediated by CB2R, in addition to acting as a neuropeptide in the brain. 9 Effects of Hp and related peptides within the vascular system 9.1 Hypotensive effect of Hp The peptide (r)Hp is named for causing a transient reduction in blood pressure in anesthetized male Wistar rats

17

(Table 3). But, compared with BK, the effect of (r)Hp is 10–100-fold lower when administered in the same

manner 69. In addition, the effect does not differ between intra-arterial (i.a.) and intravenous (i.v.) administration. Moreover, the effect can be potentiated by bacterial lipopolysaccharide instead of the BK2 receptor antagonist HOE 140 and the ACE competitive inhibitor captopril 69, suggesting that the blood pressure-lowering mechanism of Hp is either different from that of BK or independent of ACE depletion. Because lipopolysaccharide treatment elevates the level of endogenous RVD-Hpα which can inhibit NE expression 50,51, it is possible that endogenous RVD-Hpα is involved in blood pressure reduction by Hp. Besides of endogenous RVD-Hpα, endogenous nitric oxide also involves in the slight hypotensive effect of (r)Hp. It is supported by the fact that an i.v. bolus administration of (r)Hp to an anesthetized rat slightly decreased the systemic arterial pressure within 2–3 min, which was abolished by the inhibitor of nitric oxide synthesis, L-nitroarginine methyl ester 70.

11

Journal Pre-proof 9.2 Effects of Hp truncated derivatives and extended peptides on blood pressure (r)Hp(1–7) fragment (PVNFKFL) has an antinociceptive effect similar to that of (r)Hp, but it insufficiently decreased blood pressure in rabbits and rats 69, indicating that Ser8 and His9 at the C-terminus are required for (r)Hp to decrease blood pressure. VD-Hpα is reported to be a CB1R agonist 18. The intrathecal (i.t.) injection of (m)VD-Hpα (5–30 nmol) can decrease the mean arterial pressure (MAP) to a level similar to that by WIN55212-2 (1.25–10 nmol) 30. The hypotensive effect of (m)VD-Hpα appears to be exhibited through a non-CB1R, non-CB2R, and non-NO mechanism because neither the CB1R antagonist AM251 nor the CB2R antagonist AM630 as well as pretreatment with i.v. L-nitroarginine methyl ester has been reported to alter the effect of (m)VD-Hpα. Interestingly, spinal hypotension induced by (m)VD-Hpα significantly decreases after pretreatment with the αadrenoceptor antagonist phentolamine but not by the β-adrenoceptor antagonist propranolol or the muscarinic receptor antagonist atropine, indicating that α-adrenoceptor is involved in the hypotensive effect of (m)VD-Hpα. Remarkably, later studies have demonstrated that RVD-Hpα inhibits the expression of NE, which increases blood

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pressure 50,51. Due to the similar pharmacological characteristics to RVD-Hpα, it is possible that VD-Hpα decreases blood pressure via a mechanism of reduction NE expression. Thus, pretreatment with phentolamine has occupied the α-adrenoceptor on vascular smooth muscle cells and leads to dilate vessels, thereby the effect of VDHpα cannot be displayed. Additionally, it is possible that the CB1R agonist (m)VD-Hpα decreases mean arterial pressure through a mechanism similar to that of anandamide or WIN55212-2 which accounts for vasorelaxation in vascular smooth muscle cells through reduction in Ca2+ influx via the L-type channel 8; however, this still needs to

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be clarified.

Table 3. Effects of Hp and its related peptide on blood pressure

(r)Hp

Dose (nmol/kg) *

Pr

Animals

Administration

Male Wistar rats

i.v.

al

Peptides

0.0009, 0.009,

Direction of

Reference

change ↓a

17

0.09, 0.9–9

i.v., i.a.

1, 10, 100

↓

69

i.v., i.a.

1, 10, 100

↓

69

Male C57/BL6 mice

i.v., i.a.

1, 10, 100

↓

69

Male New Zealand

i.v., i.a.

100–1000

−b

69

i.t.

25–150

↓

30

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Male New Zealand white rabbits

(r)Hp(1–7)

Jo

Male Wistar Rats

white rabbits (m)VD-

Male Wistar rats

Hpα *, a

dose of per kg body weight

↓, decrease

b −,

no change

10 CBR-mediated effects of Hp and related peptides 10.1 Antinociceptive effect of VD-Hpα The antinociceptive effects of VD-Hpα are summarized in Table 4. As a CB1R agonist, (m)VD-Hpα has a dose-dependent antinociceptive effect at the levels of spinal cord and above in the tail flick test 71. The effect was 12

Journal Pre-proof blocked by CB1R antagonist AM251 but not CB2R antagonist AM630 or naloxone, suggesting that the mechanism is related to CB1R instead of CB2R or opioid receptors 71. The supraspinal analgesia induced by (m)VD-Hpα is reduced by CB1R neutral antagonist AM4113 in the mouse hind paw formalin test, skin incision-induced postoperative pain assay and acetic acid-induced visceral pain model 72. Moreover, the transient receptor potential vanilloid type 1 (TRPV1) receptor is also involved on the analgesia of (m)VD-Hpα in the acetic acid-induced visceral pain model, for that analgesia could be reduced by TRPV1 receptor antagonist SB366791 72. (r)VD-Hpα induces the same antinociceptive effect as those observed for (m)VD-Hpα via i.t. or intracerebroventricular (i.c.v.) administration 72,73. Together, VD-Hpα possesses antinociceptive effects mediated by CB1R. Table 4. Effects of VD-Hpα on pain Peptide

Pain model

Administration

Dose

Pain

Reference

(nmol) * Tail flick test

i.c.v.

3.75–22.5

↓a

73

Tail flick test

i.t.

1.88–15

↓

73

Tail flick test

i.c.v.

3.75–22.5

↓

71

i.t.

1.8–15

↓

71

i.c.v.

5–20

↓

72

i.c.v.

5–20

↓

72

Hind paw formalin test II

i.c.v.

5–20

−b

72

Acetic acid-induced writhing test

i.c.v.

5–20

↓

72

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(r) VDHpα

(m)VD-

Tail flick test

pain assay

*,

ur n

al

Hind paw formalin test I

Pr

Skin incision-induced postoperative

e-

Hpα

total dose of per injection decrease

b −,

no change

Jo

a ↓,

10.2 Neuromodulation

The neuromodulatory effect of Hp and related peptides has been investigated in neural stem cells and Neuro2A cells (Table 5). Neural stem cells in the subventricular zone have the abilities of self-renewing; differentiating; and producing neurons, astrocytes, and oligodendrocytes. In vitro treatment with Hp promotes differentiation and maturation of neural stem cells in the subventricular zone without any effects on cell apoptosis and proliferation 58. Neuro-2A cells are a mouse neuroblastoma cell line with neuronal and amoeboid stem cell morphology. Hp blocks agonist-mediated increase in Neuro-2A cells expressing CB1Rs in a manner similar to that for SR141716 29. In contrast, RVD-Hpα and VD-Hpα promote neurite outgrowth in a manner similar to that of the CB1R agonist Hu210 in Neuro-2A cells; this effect could be inhibited by pretreatment with the antagonist SR141716 18. (m)VDHpα and (r)VD-Hpα, differing in terms of one amino acid, have the similar ability to promote significant neurite outgrowth in Neuro-2A cells 73. Overall, Hp and extended peptides RVD-Hpα and VD-Hpα could cause CB1Rmediated neuromodulation. Table 5. Effects of Hp and related peptides on neurite outgrowth 13

Journal Pre-proof Peptide

Cells

Dose (μM)

Effects on neurite outgrowth

Reference

(r)Hp

Neuro-2A

1

↓a

29

0.1 or 1

Promotes differentiation and maturation

58

cells Neural stem cells

but without impacts on cell apoptosis and proliferation

(m)VD-Hpα

Neuro-2A

↑b

1

18

cells

(r)VD-Hpα

73

Neuro-2A

1

↑

1

↑

73

Neuro-2A

Hpα

cells

a ↓,

decrease

b ↑,

increase

18

e-

(m)RVD-

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cells

10.3 Appetite reduction

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Like CB1R antagonist SR141716 74, Hp also exhibits an appetite suppressant effect on the brain (Table 6). A study conducted in 2010 showed that Hp administration by i.c.v or i.p. injection dose dependently reduces nocturnal food intake in normal male rats and mice as well as in obese male ob/ob mice 75. The hypophagic effect 75.

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of (m)Hp was demonstrated to be mediated by CB1R in vivo because the effect of Hp disappears in CB1R−/− mice Moreover, (m)Hp inhibits WIN55212-2-induced eGFP-CB1R internalization and CP55940-induced

hyperphagia 75. Remarkably, compared with rimonabant and AM251 (its derivative), which reduce food intake and

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lead to excessive scratching, no adverse effects have been found for Hp in behavioral studies 75. However, this is not sufficient to support the effect of (m)Hp on appetite. It is necessary to demonstrate if (m)Hp has unnecessary and excessive impacts on the central reward route. The data on c-Fos protein immunohistochemistry and magnetic

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resonance imaging revealed that the i.p. injection of (m)Hp primarily activates feeding-related circuits of the mediobasal hypothalamus and nociceptive regions of the periaqueductal gray and dorsal raphe nucleus without the activation of brain reward centers such as the ventral tegmental area, nucleus accumbens, and orbitofrontal cortex 56.

This indicates that the systemic administration of (m)Hp selectively acts on pain and appetite without activating

the central reward route. Table 6. Effects of Hp and its related peptide on appetite Peptide

Animals model

Administration

Dose

Appetite

Reference

(nmol) * (m)Hp

Male CD1 mice

i.p.

500

↓a

75

Male CD1 mice

i.c.v.

1, 5, 10

↓

75

Male Sprague–Dawley rats

i.c.v.

10

↓

75

14

Journal Pre-proof i.p.

500

↓

75

Male CB1−/− mice

i.p.

500

−b

75

Male Kunming mice

i.c.v.

6.7, 13.4,

↑c

71

↓

51

Homozygous leptin-deficient (ob/ob) mice

(m)VD-Hpα

20.1 (m)RVD-

Adult male Sprague–Dawley rats

i.c.v.

10

Hpα i.c.v., total dose of per injection; i.p., dose of per kg body weight

a ↓,

decrease

b −,

no change

c ↑,

increase

10.4 Inhibition of gastrointestinal (GI) motility

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*

In the gut, CB1R is primarily distributed in the myenteric and submucosal ganglionated plexuses of the ileum and colon 7. CB1R is shown to modulate GI function 76,77. As CBR-binding proteins, the i.c.v. administration of

e-

(m)Hp or (m)VD-Hpα inhibits upper GI transit and colonic expulsion. The effects of (m)Hp and (m)VD-Hpα are prevented by AM251 instead of AM630, suggesting that (m)Hp and (m)VD-Hpα inhibit GI transit via the

Pr

activation of CB1R located in the brain 78. However, it is still unclear that why as the dose increases, (m)Hp tends to delay the whole gut transit, whereas (m)VD-Hpα does not function. In addition, it still needs to clarify that the effect of the per os (p.o.) administration of (m)Hp, an orally active inhibitor of CB1R, on GI transit. Moreover,

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little is known about the effects of Hp truncated derivatives and other extended peptides on GI transit. 10.5 Alleviation of liver fibrosis

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In the normal liver, the expressions of CB1R and CB2R are low or absent 79. CB2Rs are shown to upregulate in liver cirrhosis 80 as well as alcoholic and nonalcoholic liver diseases 81,82, while relatively little is known about the function of CB1R in liver diseases. A study compared the effects of CB2R agonist β-caryophyllene and CB1R antagonist Hp on bile duct ligation-induced liver fibrosis. It was found that both β-Caryophyllene and Hp could

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alleviate liver fibrosis in the bile duct-ligated rats 83. The p.o. administration of Hp for 2 weeks could reduce hepatic collagen deposition, downregulate CB1R and CB2R expression, and increase matrix metalloproteinase-1 expression 83. Another study compared the effects of curcumin and Hp on the same liver fibrosis model. It was shown that the p.o. administration of Hp also reduced the gene expression of cyclooxygenase-2 and nuclear factorkappa B 84. 10.6 Reduction in renal reabsorption Na+/K+–ATPase is critical for Na+ reabsorption in the proximal tubule. In an in vitro study, (r)Hp increased Na+/K+–ATPase activity in a TRPV1 antagonist-sensitive manner after 1-min incubation. However, Hp decreased Na+/K+–ATPase activity after 15-min incubation in porcine proximal tubular LLC-PK1 cells. CB1R antagonist or protein kinase A inhibitor could block the effect of Hp after 30-min incubation, suggesting that the activation of CB1R and the protein kinase A signaling pathway are involved 85. In contrast to Hp, (m)RVD-Hpα or (m)VD-Hpα increased Na+/K+–ATPase activity after 30-min incubation 85. In view of the critical roles of cannabinoid system in renal functions 86, it is promising to use Hp and related peptides to modulate renal functions, thereby more further studies are needed. 10.7 Modulation of other signaling pathways 15

Journal Pre-proof Hp and its derivatives are also involved in the regulation of other signaling pathways. It has been reported that the i.c.v. injection of (r)Hp completely blocks the hyperesthesia induced by endokinin A/B and prolongs the effect of endokinin C/D 87, suggesting a modulating effect of Hp on the tachykinin system. The peptides (r)Hp and (r)Hp(1–7) could activate G protein and replace the radiolabeled μ receptor selective ligand [3H]DAMGO, which can be slightly blocked by naloxone 33, suggesting a modulating effect of (r)Hp and (r)Hp(1–7) on opioid system. Additionally, (m)VD-Hpα is shown to alter the pharmacological characteristics of neuropeptide FF 88. 11 Other activities of Hp and related peptides 11.1 Antinociceptive effect of Hp and its truncated derivatives The antinociceptive effect of Hp appears inconsistent in several models of pain (Table 7). (r)Hp effectively reduces hyperalgesia in Cg-induced or BK-induced hyperalgesia models whether it is administrated via intraplanar, i.t., or p.o. injection 29. The effect of (r)Hp is not altered by naloxone, suggesting that an opioid receptor mechanism is not involved 35. Hp also had a significant antinociceptive effect on an acetic acid-induced

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visceral pain model 29. However, (r)Hp had no effect on a Cg local injection-induced joint pain model or neuropathic spinal cord injury pain model as well as in the hot plate test 89. In the hind paw formalin test, the effect of (r)Hp appears to be determined by the administration method. The i.t. injection of (r)Hp alleviates pain, whereas its i.c.v. injection enhances pain 89. All these data indicate that (r)Hp has inexplicable variability in physical pain. It has been demonstrated that the deletion of five, but not four, amino acids from the C-terminus of Hp affects

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its activity of CB1R recognition 29. However, Hp(1–5) is as inactive as Hp(1–4) in Cg-induced hyperalgesia 35. Only Hp(1–6) and Hp(1–7) have been reported to be as effective as Hp35, suggesting that Hp(1–6) is the minimal

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sequence necessary to obtain the antinociceptive activity. Table 7. Effects of Hp on pain

Dose (μg)

Pain

Reference

i.pl

10

↓a

29,35

i.pl.

1, 10, or 20

↓

35

i.t.

0.5 or 5

↓

29

p.o.

50 or 100

↓

29

i.pl.

10

↓

35

Acetic acid-induced writhing test

i.p.

50 or 100

↓

29

Preclinical hot plate test

i.t.

10

−b

89

i.c.v.

10

−

89

i.t. (pretreatment)

0.1, 0.3, 1,

↓

89

10

−

89

3 or 10

−

89

Pain model

(r)Hp

Paw pressure test

al

Administration

Peptide

ur n

Carrageenan-induced hind paw

Jo

inflammatory hyperalgesia

Bradykinin-induced inflammatory hyperalgesia

Hind paw formalin test 1

or 3

i.c.v.

16

Journal Pre-proof Hind paw formalin test 2

3

↓

89

10

↑c

89

3

↑

89

10

↑

89

i.t.

0.1, 1, or 10

−

90

i.c.v.

0.1, 1, or 3

−

90

i.t. (after treatment)

0.3–30

−

91

↓

92

i.t. (pretreatment)

i.c.v.

Mechanical allodynia in a rat model of neuropathic pain in spinal cord injury

Mechanical allodynia in carrageenan-

(m)Hp

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induced osteoarthritis pain model Tail flick test

i.c.v.

12, 24, or 49

i.pl., dose of per paw; i.c.v. or i.t., total dose of per injection; i.p., dose of per kg body weight decrease

b −,

no change

c ↑,

increase

Pr

a ↓,

e-

*

11.2 Inducing tolerance to thermal antinociception

Drug tolerance is a pharmacological concept indicating subjects’ reduced response to a drug following its repeated use. Cross-tolerance is a phenomenon that occurs when tolerance to the effects of a certain drug produces

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tolerance to the effects of another drug. It is well known that cannabinoid agonists induce the development of tolerance 93,94. Accordingly, (m)Hp and (m)VD-Hpα have been demonstrated to induce tolerance in a radiant heat

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tail flick test after repeated i.c.v. administration for 4 days 71,92. Moreover, cross-tolerance has been shown between (m)Hp and (m)VD-Hpα 92. Although (m)Hp and (m)VD-Hpα are CB1R antagonist and agonist, respectively, they have similar characteristics in inducing tolerance to thermal antinociception in addition to

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eliciting hypotension. (r)VD-Hpα, different in one amino acid from (m)VD-Hpα, induced tolerance on day 3, a day faster than (m)VD-Hpα 73.

11.3 Conditioned place aversion Conditioned place preference (CPP) can be used to measure the neural circuits involved in drug reward. Cannabinoids have complex effects on the conditioned place preference paradigm. Cannabinoids can induce CPP and conditioned place aversion depending on variations in procedural details across laboratories 95. The i.c.v administration of (m)VD-Hpα has been reported to produce a response of conditioned place aversion using a CPP apparatus with three compartments 71. 11.4 Effect on memory Hp and its extended peptides are also shown to affect memory. The i.c.v. administration of (m)Hp to mice before training prolongs memory retention in addition to improving memory formation. Conversely, the i.c.v. administration of (m)RVD-Hpα or (m)VD-Hpα impairs memory 96. Thus, it is easy to understand that the i.c.v. administration of (m)RVD-Hpα or (m)VD-Hpα at the same dose can prevent the effect of the i.c.v. administration of (m)Hp, whereas the i.c.v. administration of (m)Hp can block the effect of i.c.v. (m)RVD-Hpα or (m)VD-Hpα 96. The effects of (m)Hp and (m)RVD-Hpα or (m)VD-Hpα are also inconsistent in amyloid-β (1–42) (Aβ1–42)17

Journal Pre-proof induced memory impairment in mice. Hp improves memory in normal mice, but is inactive in Aβ1–42-induced memory impairment in mice. (m)RVD-Hpα or (m)VD-Hpα impairs memory in normal mice, but they can reverse Aβ1–42-induced memory impairment in mice 96. Of note, in addition to memory, the opposite effects of (m)Hp and its extended peptides are also shown in modulation of appetite and anxiety. 11.5 Effect on anxiety and depression The effects of Hp and related peptides on anxiety and depression are shown in Table 8. Regarding of the use of i.p., i.c.v. or p.o. administration, the anxiety-inducing effect of (r)Hp was always observed. However, the mechanism of inducing anxiety differed among the administration routes. In i.p. administration of Hp, intact Hp couldn't be detected in brain extracts by mass spectrometry, suggesting inability of intact Hp to cross the BBB, thereby the anxiety-inducing effect might be attributed to Hp fragment or other fragments 55. In i.c.v. administration of Hp, the effect was prevented by TRPV1 antagonists instead of the CB1R antagonist AM251, suggesting that Hp induces anxiety via activating TRPV1 receptors 55. Because of the BBB, the activity of intact

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(r)Hp by p.o. administration may also the result of Hp fragment or other fragments 55. Moreover, a single i.p. injection of (m)Hp induced behavioral despair in the forced swim test 97, suggesting that (m)Hp induces depression.

Contrary to Hp, RVD-Hpα induces anxiolytic and antidepressive effects (Table 8). A single i.p. injection of RVD-Hpα decreases anxiety-related behavior and total immobility in rats without modifying locomotion, which is

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involved the modulatory effects of RVD-Hpα on monoaminergic signaling in the prefrontal cortex 97. After treatment with RVD-Hpα, in the prefrontal cortex, the levels of dopamine, NE, and serotonin, which are critical in

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locomotion and emotional behavior, increased 97, and meantime, the expressions of monoamine oxidase and catechol-O-methyltransferase gene, which are the main enzymes involved in the catabolism of catecholamines and serotonin, decreased 97. Similar to the i.p. injection of RVD-Hpα, its i.c.v. administration resulted in anxiolytic

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behavior in rats, which is associated with decrease in NE levels and orexin-A gene expression in the hypothalamus 51.

(r)Hp

Models

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Peptide

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Table 8. Effects of Hp and related peptides on anxiety and depression Administration

Dose

Anxiety

Depression

Reference

(μg) *

Elevated plus maze test

i.p.

50

↑a

55

Elevated plus maze test

i.c.v.

3.3,

↑

55

10.9

(m)Hp

(m)RVD-

Elevated plus maze test

p.o.

500

↑

55

Open-field test

i.p.

50

↑

97

Light–dark exploration test

i.p.

50

↑

97

Forced swim test

i.p.

50

Open-field test

i.p.

50

↓b

97

Light–dark exploration test

i.p.

50

↓

97

↑

97

Hpα

18

Journal Pre-proof Forced swim test

i.p.

50

Open-field test and elevated

i.c.v.

10.9

↓ ↓

97

51

plus maze test *

i.p. and p.o., dose of per kg body weight; i.c.v., total dose of per injection

a ↑,

increase

b ↓,

decrease

11.6 Effect of extended peptides of Hp on appetite VD-Hpα, functions like CB1R agonist, has the consistent effects with Hp on blood pressure and pain, but displays inconsistent effect to Hp on appetite (Table 6). Unlike Hp, the i.c.v. injection of (m)VD-Hpα causes a dose-dependent increase in food intake in mice 71. RVD-Hpα, a PAM of CB2R in addition to a NAM of CB1R,

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inhibits food consumption without an impact on the total body weight after14 daily i.p. injections 51. But, the mechanism of CBR mediation remains to be determined because only the reduction of hypothalamic NE levels as well as proopiomelanocortin gene and orexin-A gene expressions is reported 51. Moreover, it is still necessary to uncover the effects of C-terminally truncated derivatives of Hp and other pepcans on appetite. 11.7. Effect on the adrenal glands

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Abundant (m)RVD-Hpα has been reported in both nonperfused and perfused adrenal glands 23. After adrenalectomy, the levels of (m)RVD-Hpα in the brain, liver, kidneys, spleen and lungs significantly reduced 24,

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suggesting that the adrenal glands are the major sites of (m)RVD-Hpα presence/production. The adrenal medulla is the critical organ to secrete catecholamine (NE and adrenaline) which is modulated by CB1R 98. As an allosteric modulator of CBRs, pretreatment with i.c.v. injection of (m)RVD-Hpα, instead of (r)Hp, inhibits the expression of plasma catecholamines induced by the i.c.v. administration of bombesin (a stress-related neuropeptide) in rats,

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suggesting that activation but not inhibition of CB1R in the brain inhibits central adrenomedullary outflow 50.

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12 Outlook

In view of the pharmacological activities of Hp and related peptides, it is promising to use them to modulate numerous physiological and pathological processes which is involving cannabinoid system. However, before considering their application, it is critical to take into account two of their properties: easy hydrolysis in tissues and

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blood, and inability to cross the BBB, thereby using them with proper indications, sites, and administrations. Regarding antinociception, although Hp, its truncated derivatives, and extended peptides were shown to be effective, the effects of VD-Hpα were more stable than Hp via i.c.v and i.t. administration. Moreover, as it is easily hydrolyzed, Hp and related peptides are suitable to be locally administrated to reduce pain. Regarding neuromodulation, RVD-Hpα is preferable over Hp because it is more stable. But the caution is needed when used to treat Alzheimer’s disease for that RVD-Hpα self-assembles at high concentrations, and impairs memory via the i.c.v. administration. It appears that Hp and RVD-Hpα are suitable to treat hyperphagia and obesity because of few adverse effects. Especially, RVD-Hpα appears to be better, because RVD-Hpα reduced anxiety and depression but Hp induced them. In addition, it should be noted that although many studies involved Hp, VD-Hpα, and RVDHpα, it appears that RVD-Hpα and its precursor pepcan-23 are the only peptides detected at the physiological level 24.

Thus, further in-depth studies on Hp and its derivatives are still necessary for future drug development.

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Journal Pre-proof Acknowledgments Funding: This work was supported by the Special Fund of the Chinese Central Government for Basic Scientific Research Operations [lzujbky-2017-129]. Declarations of interest

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None.

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Leone S, Recinella L, Chiavaroli A, et al. Emotional disorders induced by Hemopressin and RVD-

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Journal Pre-proof Highlights

Cannabinoid receptors (CBRs) are involved in various processes, e.g., nociception.

Hemopressin acts as a CB1R inverse agonist.

RVD-hemopressin acts in a manner consistent with allosteric modulators of CBRs.

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It is appealing to use hemopressin and related peptides for therapeutic purposes.

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