The influence of μ-opioid receptor agonist and antagonist peptides on peripheral blood mononuclear cells (PBMCs)

doi:10.1016/j.peptides.2010.12.003

Peptides

Volume 32, Issue 4, April 2011, Pages 707-712

https://doi.org/10.1016/j.peptides.2010.12.003 Get rights and content

Abstract

Milk is one of the main source of biologically-active peptides that may function as regulatory substances called food hormones. After passing the gut–blood barrier, the μ-opioid receptor agonist and antagonist peptides may become the new factors influencing various functions of the human organism. The aim of the conducted research was to determine the influence of μ-opioid receptor agonist peptides: human and bovine β-casomorphin-7 (h/bBCM-7) and antagonistic peptides: casoxin-6 and- D (CXN-6/D) on proliferation and cytokine secretion of human peripheral blood mononuclear cells (PBMCs). The PBMCs proliferation was measured by the use of the BrdU test, which assesses the DNA synthesis activity and the WST-1 test which assesses the activity of mitochondrial dehydrogenase enzymes. The influence of all the investigated peptides on secretion of IL-4, IL-8, IL-13 and IFN-γ was determined by the use of the ELISA tests. Incubating the cells with the peptides has not caused any changes to their enzymatic activity, which has been proved by a WST-1 test. When using a BrdU test, however, it has been observed that there appear changes to proliferation of PBMCs correlated to amounts of bromodeoxyuridine incorporated into the cellular DNA. Moreover, changes to secretion of IL-4 and IL-13 by the cells under the influence of agonists were detected, as well as changes to secretion of IFN-gamma under the influence of all the examined substances. The obtained results provide information on immunomodulatory effects of food-derived opioid peptides, which may be of clinical significance especially in the case of allergic diseases in newborns.

Research highlights

▶ All examined peptides caused no significant changes to PBMCs proliferation measured by the enzymatic activity of the cells in the long-term incubation. ▶ Amounts of incorporated BrdU reveal a stimulation of PBMCs proliferation by bovine and human β-casomorphin-7, which indicates a short-term influence of opioid peptides on PBMCs. ▶ The μ-opioid receptor agonist peptides were found to cause a significant increase in IL-4 secretion, an insignificant increase in IL-13, and a decrease of IFN-γ secretion, which suggest an activation of the Th2 pathway.

Introduction

Milk proteins, both bovine and human, make a rich source of biologically active peptides. The μ-opioid receptor agonist peptides, called β-casomorphins (BCMs), are released from the β-casein [37], [13] and the antagonist peptides, called casoxins (CXNs), are released from the κ-casein and α_s1-casein [39], [40]. Those peptides have been identified both in cow [36] and human's milk [30], [10] as well as in numerous dairy products [4], [31]. They may be liberated from native milk proteins as a result of enzymatic hydrolysis occurring during technological processing of food [20] or in the digestive tract [5], [3]. A possibility of crossing the intestinal barrier by the opioid peptides has also been reported [9], [32] which has been confirmed by clinical research by Kost et al. [12].

The cells of the digestive, immune, nervous, and endocrine systems are equipped with opioid receptors that modulate functioning of the organism by the agency of endogenous opioids [19]. It has been suggested that upon getting into the bloodstream the exogenous opioid peptides may modulate functioning of the mentioned systems by interactions with μ-opioid receptors [34], [8]. Some authors suggest the regulatory influence of β-casomorphins on the neonates’ development [38], yet on the other hand, β-casomorphins liberated from the cow's β-casein are accused of participating in etiology of such conditions as: autism [24], [33], SIDS [34], diabetes type I, postpartum psychosis [17], circulatory disorders, or food allergies [15], [16]. Due to those facts, an increasing number of organizations, including the European Economic Community, the European Union, and the European Food Safety Authority have become involved in regulations connected with the safety of consumption of biologically active peptides in food [22].

Therefore, it is important to define the dose-dependent influence of opioid peptides consumed by people on the functioning of the human body. Moreover, the fact that both peptides, the μ-opioid receptor agonists and its antagonists, coexist in consumed food motivates to search for dependencies resulting from the possibility of simultaneous influence of both those compounds on the human organism.

The aim of this research was to examine the influence of μ-opioid agonist (bovine and human β-casomorphin-7) and antagonist peptides (casoxin-6 and casoxin-D) derived from bovine and human milk proteins on proliferation and cytokine's secretion by PBMCs.

Section snippets

Study participants

The examined material consisted of the blood obtained from five selected healthy male volunteers aged 25–30 years old with no symptoms of allergies. The study was conducted with an approval of the local Ethical Committee and all of its volunteers signed an informed consent and filled in a questionnaire on their health condition, which assessed such factors as their prevalence of allergic disease, past diseases and pharmacologic treatment. In the sera of all the five participants the IgE against

Assessment of proliferation changes for human peripherial blood mononuclear cells under the influence of opioid peptides derived from the bovine and human milk

The PBMCs proliferation after 12 h incubation together with the examined substances has been determined using two tests with different working spectra. The WST-1 test was used to assess the level of the mitochondrial dehydrogenase activity which is correlated with the general number of cells and their ability to proliferate. The BrdU-test-based determination of the cellular proliferation made it possible to assess the proliferation changes in time by measuring the incorporation of

Discussion

It has been proved that the cells of the nervous and immune systems are capable of communicating with each other by the agency of endogenous opioid peptides. As early as in 1986, Shavit et al. paid their attention at the new function of endogenous opioids as particles carrying information to the cells of the immune system [29]. Those cells are equipped with all three classes of opioid receptors (μ, δ, and κ) involved in transmitting signals by endo- and exogenous opioids [18]. Moreover,

Acknowledgment

The research has been conducted under project financed by the Ministry of Science and Higher Education, number NN407 153939.

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The influence of μ-opioid receptor agonist and antagonist peptides on peripheral blood mononuclear cells (PBMCs)

Abstract

Research highlights

Introduction

Section snippets

Study participants

Assessment of proliferation changes for human peripherial blood mononuclear cells under the influence of opioid peptides derived from the bovine and human milk

Discussion

Acknowledgment

Food Chem

Peptides

Peptides

FEBS Lett

Peptides

J Biol Chem

Rev Allergol

Drug Alcohol Depend

J Dairy Sci

Int Dairy J

Neuroscience

Int Dairy J

Int Dairy J

Peptides

J Allergy Clin Immunol

Peptides

Livestock Prod Sci

Human b-casomorphin-5 containing peptides in human body fluids

Immune cell-derived beta-endorphin. Production, release, and control of inflammatory pain in rats

J Clin Invest

Beta-casomorphin 7 in raw and hydrolyzed milk derived from cows of alternative β-casein genotypes

Milchwissenschaft