Bradykinin stimulates prostaglandin E2 release in human skeletal muscular fibroblasts

https://doi.org/10.1016/j.mce.2020.110771Get rights and content

Highlights

  • Bradykinin stimulated PGE2 release through bradykinin B2 receptors, in human fibroblasts obtained from skeletal muscle.

  • Bradykinin induced ERK1/2 and p38 phosphorylation and provoked the translocation from the cytosol to the nucleus of p65/NF-kB.

  • P65/NF-kB activation induced the increase of COX-2 expression and subsequent PGE2 synthesis.

  • The release of PGE2 provide a possible explanation for the role of bradykinin in inflammatory diseases, such as DOMS.

Abstract

Local mediator prostaglandins and bradykinin are involved in inflammation and pain. We explored bradykinin effects on prostaglandin E2 (PGE2) release from fibroblasts derived from human skeletal muscular biopsies. Bradykinin induced PGE2 release through bradykinin B2 receptors, since PGE2 release was blocked by the bradykinin B2 receptor selective antagonist FR173657 and B2 receptor agonist (Hyp3)-bradykinin showed effects comparable to bradykinin. Consistently, bradykinin induced both mRNA cyclooxygenase 2 (COX-2) and protein. Bradykinin also induced ERK1/2 and p38 phosphorylation and provoked the translocation from the cytosol to the nucleus of p65/NF-kB. The release of PGE2 by bradykinin could be blocked inhibiting COX-2 and p65/NF-kB, ERK1/2 or p38 activation. Both ERK1/2 and p38 were upstream to NF-kB inasmuch siRNAs significantly blocked the p65/NF-kB activation induced by bradykinin. Thus, bradykinin, acting via B2 receptors, induced PGE2 release through ERK1/2 and p38-dependent pathways and consequent p65/NF-kB translocation to nucleus. p65/NF-kB induced COX-2 transcription. The release of PGE2 provide a possible explanation for the role of bradykinin in inflammatory diseases.

Introduction

The symptoms of delayed onset muscle soreness (DOMS) are usually observed following heavy and atypical exercise (Armstrong, 1984). Several hypotheses may explain the mechanism of DOMS such as: lactic acid, connective tissue damage, muscle damage, inflammation, and oxidative stress (Armstrong, 1984; Smith, 1991; Cheung et al., 2003; Malm et al., 2004; Crameri et al., 2007). During muscle exercise many mediators are released; these include bradykinin (including kallidin-like peptide) (Blais et al., 1999; Boix et al., 2002), ATP and glutamate (Li et al., 2003). These substances are able to induce not only excitation/sensitization of nociceptors, but also changes in expression of neuropeptides and channels in several types of cell (Ferreira et al., 1993; Baker, 2005; Inoue et al., 2006).

It is known that bradykinin is able to cause various biological effects; these include the contraction of smooth muscle and vasodilation, bronchoconstriction, inflammation, pain and edema and, not least, cell proliferation (Liebmann, 2001). Bradykinin acts through two receptors called B1 and B2 and it is commonly accepted that a large part of its physiological actions are mediated by B2 receptors (Hall, 1992).

Bradykinin released during exercise plays an essential role, through B2 receptors, in the muscle mechanical hyperalgesia (Murase et al., 2010). Bradykinin is able to stimulate the release of prostaglandins in guinea-pig isolated lungs (Palmer et al., 1973), in dog's knee joint (Ferreira et al., 1974; Moncada et al., 1975), in kidney (McGiff et al., 1972) and in spleen (Ferreira et al., 1973), in the heart (Needleman et al., 1975) and ear of the rabbit (Juan and Lembeck, 1976; Lembeck et al., 1976).

Most cell types including epithelial cells, fibroblasts, and macrophages, are capable of producing prostaglandins, which are part of the eicosanoid family (oxygenated hydrophobic derivatives of C20 fatty acids) (Smith, 1989; Berenbaum, 2000; Funk, 2001). PGE2, the most abundant prostanoid in the human body (Serhan and Levy, 2003), has been shown to have a number of biological actions, including vasodilation and both anti- and pro-inflammatory actions (Sugimoto et al., 2000; Davies et al., 1984; Takayama et al., 2002). PGE2 is a primary product of arachidonic acid metabolism in many cells; it does not exist preformed in the cell like most eicosanoids, but it is synthesized de novo and released into the extracellular space after cell activation or supply of exogenous free arachidonate. PGE2 is derived from PGH2 which in turn is synthesized from arachidonic acid through the cyclooxygenase (COX) pathway (Park et al., 2006).

A recent human study has supported PGs involvement in DOMS and muscle performance as the non-steroidal anti-inflammatory drug ibuprofen was able to diminish both DOMS and the drop of muscle performance after strenuous eccentric contractions (Headly et al., 1986). Therefore, it is important to understand factors and conditions that might affect the PGE2/synthesis. Thus, in this paper we aimed to examine the release of PGE2 in fibroblasts obtained from skeletal muscular biopsies stimulated by bradykinin, and the intracellular mechanisms activated by bradykinin in these cells.

Section snippets

Materials

Antibiotics, glutamine, foetal bovine serum (FBS) and anti- α-actin antibody were purchased from Celbio (Pero, MI, Italy). Anti-phosphorylated and unphosphorylated ERK1/2 and p38 antibodies, anti-p65, IkB-α, bradykinin B2 receptor, bradykinin B1-receptor and goat anti-rabbit and donkey anti-goat conjugated with peroxidase, as well as control antibodies, were obtained from Santa Cruz Biotechnology (USA).

SB203580 and PD98059 were obtained from Calbiochem (San Diego, CA, USA). Recombinant human

Effect of bradykinin on the production of PGE2

FACS analysis of the initial (pre-enrichment) cells after 7 days culture showed distinct myogenic (desmin) and fibroblast populations (TE-7), which accounted for virtually all of the adherent muscle-derived population (Fig. 1A). The TE-7 fibroblast marker was the most discriminating for fibroblasts and was completely absent from myogenic cells (Agley et al., 2013). After fibroblast enrichment, about 95% of cell in primary culture expressed TE-7, at least until the third passages (Fig. 1A).

B1

Discussion

Bradykinin and prostaglandins are both local mediators strongly implicated in pain and inflammation. Bradykinin is a peptide with powerful physiological actions; it is associated with conditions such as sepsis, immune-mediated lesion, DOMS, multiple trauma and is involved in acute lung injury and inflammatory response (Blais et al., 1999; Boix et al., 2002; Pixley et al., 1995). It has previously been shown that bradykinin stimulated PGE2 release in several cell types, such as skin and foreskin

Funding

This study was supported by Italian Ministry of Education, University and Research (MIUR) grant number FFABR_ANVUR_20/2018.

CRediT authorship contribution statement

Antonella Muscella: Conceptualization, Writing - original draft. Luca Giulio Cossa: Data curation, Software, Methodology. Carla Vetrugno: Data curation, Formal analysis. Santo Marsigliante: Writing - review & editing, Supervision.

Declaration of competing interest

The authors have no conflict of interest to declare.

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