Dynorphin A 1–17 biotransformation in inflamed tissue, serum and trypsin solution analysed by liquid chromatography–tandem mass spectrometry

Abstract

Dynorphin A 1-17 (DYN A) is an endogenous neuropeptide that is of interest due to its diverse roles in analgesia, inflammation and addiction. Upon release, DYN A is subject to metabolism by a range of enzymes and its biotransformation is dependent on the site and environment into which it is released. In this study, we investigated the biotransformation of DYN A in rat inflamed tissue at pH 7.4 and 5.5, in rat serum and in trypsin solution. DYN A-porcine was incubated at 37 °C in each matrix over a range of incubation periods. The resultant fragments were separated using a C4 column and detected by mass spectrometry using total ion current mode. Incubation of DYN A in trypsin solution and in rat serum resulted in 6 and 14 fragments, respectively. Incubation in inflamed rat paw tissue occasioned 21 fragments at pH 7.4 and 31 fragments at pH 5.5. Secondary breakdown of some larger primary fragments was also observed in this study.

Inhibitory effects of dynorphin 3-14 on the lipopolysaccharide-induced toll-like receptor 4 signalling pathway

“…Consistent with the abundant local accumulation of DYN 3-14 following biotransformation of DYN 1-17 in rodent inflamed tissue [6], this study has described a potential role for DYN 3-14 as an endogenous immune modulator in the tissue through modulation of pro-inflammatory cytokine release and the action upon TLR4-mediated NF-κB/p65 pathways.…”

“…In congruence with the lower potency exhibited by DYN 3-14, this peptide was produced abundantly following biotransformation of DYN 1-17 in rodent inflamed tissue homogenate [6]. Furthermore, DYN 3-14 does not undergo rapid degradation (93.40% remaining after 1 h, data not shown) upon incubation with THP-1 cells, suggesting that high local concentrations are present in the inflammatory milieu.…”

“…Biotransformation resulting in the removal of the aminoterminal tyrosine (Tyr) residue in opioid peptides yields fragments that are structurally "nonopioid" in nature with very low affinity for opioid receptors [26]. The immunoregulation observed may be facilitated by a range of opioid and non-opioid fragments, produced following biotransformation of DYN 1-17 at the site of inflammation [6]. Outside of traditional opioid receptor-mediated effects, evidence of non-opioid receptors roles for opioid peptides include action at melanocortin receptors [27,28], bradykinin receptors [29], Nmethyl-D-aspartate (NMDA) receptor complex [30] and recently the TLR4 [20], therefore the overarching effects of biotransformed opioid peptides may involve a number of possible mechanisms.…”

“…Our previous studies have demonstrated that DYN 1-17 undergoes biotransformation in rodent inflamed tissue, resulting in a unique set of opioid and non-opioid fragments [6]. and its N-terminal biotransformation fragments have been shown to be involved in the modulation of LPSinduced NF-κB/p65 nuclear translocation and the subsequent release of IL-1β and TNF-α in THP-1 cell-derived macrophages through the kappa-opioid receptor (KOR) pathway [7].…”

The efficacy of Dynorphin fragments at the κ, μ and δ opioid receptor in transfected HEK cells and in an animal model of unilateral peripheral inflammation

“…Our group has demonstrated that dynorphin A 1-17 undergoes extensive and rapid biotransformation in an inflamed rat paw tissue homogenate, producing a unique set of dynorphin fragments [7]. It is likely that a similar set of fragments is produced from dynorphin released from leukocytes into inflamed tissue and that these dynorphin fragments mediate pain and inflammation.…”