Spinorphin

Spinorphin is an endogenous inhibitor of enkephalin-degrading enzymes (aminopeptidase, dipeptidyl aminopeptidase III, angiotensin-converting enzyme and enkephalinase) [1]. Also, it is a potent antagonist of human P2X3 receptor and the N-formylpeptide receptor subtype FPR with IC50 value of 8.3 pM for P2X3 receptor[2][3].

Enkephalin degrading enzymes are a series of enzymes that hydrolyze enkephalins, which play an important role in management of blood pressure, pain, hypertension and cardiovascular diseases.

In human embryonic kidney (HEK) 293 cells transfected with mouse FPR, spinorphin induced a typical rise in the intracellular Ca2+ concentration with EC50 of 128 μM. Also, in normal mouse neutrophils, Spinorphin induced calcium flux in a dose-dependent way. While the neutrophils from mice deficient in the fMLF receptor subtype FPR didn’t response [3].

In mice, intrathecal administration of spinorphin inhibited the allodynia induced by intrathecal nociceptin in a dose-dependent way. Furthermore, spinorphin enhanced the inhibitory effect of enkephalin on allodynia induced by nociceptin [4].

References:
[1].  Nishimura K, Hazato T. Isolation and identification of an endogenous inhibitor of enkephalin-degrading enzymes from bovine spinal cord. Biochem Biophys Res Commun, 1993, 194(2): 713-719.
[2].  Jung KY, Moon HD, Lee GE, et al. Structure-activity relationship studies of spinorphin as a potent and selective human P2X(3) receptor antagonist. J Med Chem, 2007, 50(18): 4543-4547.
[3].  Liang TS, Gao JL, Fatemi O, et al. The endogenous opioid spinorphin blocks fMet-Leu-Phe-induced neutrophil chemotaxis by acting as a specific antagonist at the N-formylpeptide receptor subtype FPR. J Immunol, 2001, 167(11): 6609-6614.
[4].  Honda M, Okutsu H, Matsuura T, et al. Spinorphin, an endogenous inhibitor of enkephalin-degrading enzymes, potentiates leu-enkephalin-induced anti-allodynic and antinociceptive effects in mice. Jpn J Pharmacol, 2001, 87(4): 261-267.

Structure-activity relationship studies of spinorphin as a potent and selective human P2X(3) receptor antagonist.[Pubmed:17676725]

J Med Chem. 2007 Sep 6;50(18):4543-7.

Spinorphin, an endogenous antinociceptive peptide (LVVYPWT), showed potent and non-competitive antagonism at the ATP-activated human P2X3 receptor (IC50 = 8.3 pM) in a two-electrode voltage clamp assay with recombinant human P2X3 receptors expressed in Xenopus oocytes. Single alanine substitutions from 1st to 4th amino acids and the cyclic form of LVVYPWT sustained antagonistic properties at the human P2X3 receptors, whereas the threonine to alanine substitution resulted in an enhancing effect of the agonistic activity.

Spinorphin as an endogenous inhibitor of enkephalin-degrading enzymes: roles in pain and inflammation.[Pubmed:12470213]

Curr Protein Pept Sci. 2002 Dec;3(6):587-99.

It is possible that enkephalins are involved in the pain-modulating mechanism in the spinal cord. Enkephalins, however, are short-lived, being rapidly degraded by various endogenous enzymes. Many substances that inhibit enkephalin-degradation have been investigated and it has been reported that some inhibitors (e.g. kelatorphan and RB101) alone showed anti-nociceptive activity. We found an endogenous factor that modulated enkephalin-degrading activity and purified it from bovine spinal cord based on its inhibitory activity toward enkephalin-degrading enzymes. Structural analysis revealed the factor to be Leu-Val-Val-Tyr-Pro-Trp-Thr and it was named Spinorphin. It has been found that Spinorphin inhibited the activity toward various enkephalin-degrading enzymes from monkey brain, especially dipeptidyl peptidase III (DPPIII, Ki=5.1 x 10(-7) M). Recently we reported that this inhibitor significantly inhibited bradykinin (BK)-induced nociceptive flexor responses. Importantly, the mode of inhibition to BK-responses by Spinorphin was different from the case with morphine. The morphine-induced blockade of BK-response was attenuated by pertussis toxin treatment, whereas that of Spinorphin was not. We also have reported roles for Spinorphin in inflammation. Spinorphin significantly inhibited the functions of polymorphonuclear neutrophils (PMNs) by suppressing the binding of fMLF to its receptor on PMNs. Further, this inhibitor suppressed the carrageenan-induced accumulation of PMN in mouse air pouches after intravenous administration. These results indicate that Spinorphin may be an endogenous anti-inflammatory regulator. The possible role of Spinorphin and its analog as regulators in pain and inflammation will be discussed.

Spinorphin inhibits membrane depolarization- and capsaicin-induced intracellular calcium signals in rat primary nociceptive dorsal root ganglion neurons in culture.[Pubmed:26053512]

J Recept Signal Transduct Res. 2015;35(6):550-8.

OBJECTIVE: Spinorphin is a potential endogenous antinociceptive agent although the mechanism(s) of its analgesic effect remain unknown. We conducted this study to investigate, by considering intracellular calcium concentrations as a key signal for nociceptive transmission, the effects of Spinorphin on cytoplasmic Ca(2+) ([Ca(2+)]i) transients, evoked by high-K(+) (30 mM) depolariasation or capsaicin, and to determine whether there were any differences in the effects of Spinorphin among subpopulation of cultured rat dorsal root ganglion (DRG) neurons. METHODS: DRG neurons were cultured on glass coverslips following enzymatic digestion and mechanical agitation, and loaded with the calcium sensitive dye fura-2 AM (1 microM). Intracellular calcium responses in individual DRG neurons were quantified using standard fura-2 based ratiometric calcium imaging technique. All data were analyzed by using unpaired t test, p < 0.05 defining statistical significance. RESULTS: Here we found that Spinorphin inhibited cytoplasmic Ca(2+) ([Ca(2+)]i) transients, evoked by depolarization and capsaicin selectively in medium and small cultured rat DRG neurons. Spinorphin (10-300 microM) inhibited the Ca(2+) signals in concentration dependant manner in small- and medium diameter DRG neurons. Capsaicin produced [Ca(2+)]i responses only in small- and medium-sized DRG neurons, and pre-treatment with Spinorphin significantly attenuated these [Ca(2+)]i responses. CONCLUSION: Results from this study indicates that Spinorphin significantly inhibits [Ca(2+)]i signaling, which are key for the modulation of cell membrane excitability and neurotransmitter release, preferably in nociceptive subtypes of this primary sensory neurons suggesting that peripheral site is involved in the pain modulating effect of this endogenous agent.

Spinorphin, an endogenous inhibitor of enkephalin-degrading enzymes, potentiates leu-enkephalin-induced anti-allodynic and antinociceptive effects in mice.[Pubmed:11829145]

Jpn J Pharmacol. 2001 Dec;87(4):261-7.

Spinorphin (LVVYPWT) has been isolated from the bovine spinal cord as an endogenous inhibitor of enkephalin-degrading enzymes. It has been reported that Spinorphin has an antinociceptive effect, inhibitory effect on contraction of smooth muscle and anti-inflammatory effect. In the present study, the effects of leu-enkephalin and Spinorphin on allodynia and mechanical and thermal nociceptions were examined in vivo using mice. Intrathecal (i.t.) administration of leu-enkephalin or Spinorphin inhibited the allodynia induced by intrathecal nociceptin in a dose-dependent manner. Furthermore, Spinorphin enhanced the inhibitory effect of enkephalin on allodynia induced by nociceptin. Naloxone antagonized both inhibitory effects of leu-enkephalin and Spinorphin, suggesting that the endogenous opioidergic system can modulate allodynia. Intracerebroventricular (i.c.v.) administration of leu-enkephalin increased the nociceptive threshold of heat or mechanical stimulation to a mouse. Although i.c.v. administration of Spinorphin had no effect on the threshold of heat or mechanical stimulation, Spinorphin enhanced and prolonged the antinociceptive effect of leu-enkephalin. The enhancement of Spinorphin on the antinociception produced by leu-enkephalin was reversed by pretreatment with naloxone. From these results, it is suggested that the effects of Spinorphin on enkephalin-induced anti-allodynic and antinociceptive effects are due to inhibition of enkephalin-degrading enzymes.

The endogenous opioid spinorphin blocks fMet-Leu-Phe-induced neutrophil chemotaxis by acting as a specific antagonist at the N-formylpeptide receptor subtype FPR.[Pubmed:11714831]

J Immunol. 2001 Dec 1;167(11):6609-14.

Spinorphin is an endogenous heptapeptide (leucylvalylvalyltyrosylprolyltryptophylthreonine), first isolated from bovine spinal cord, whose sequence matches a conserved region of beta-hemoglobin. Also referred to as LVV-hemorphin-4 and a member of the nonclassical opioid hemorphin family, Spinorphin inhibits enkephalin-degrading enzymes and is analgesic. Recently, Spinorphin was reported to block neutrophil activation induced by the chemotactic N-formylpeptide N-formylmethionylleucylphenylalanine (fMLF), suggesting a potential role as an endogenous negative regulator of inflammation. Here we use both gain- and loss-of-function genetic tests to identify the specific mechanism of Spinorphin action on neutrophils. Spinorphin induced calcium flux in normal mouse neutrophils, but was inactive in neutrophils from mice genetically deficient in the fMLF receptor subtype FPR (N-formylpeptide receptor). Consistent with this, Spinorphin induced calcium flux in human embryonic kidney 293 cells transfected with mouse FPR, but had no effect on cells expressing the closely related fMLF receptor subtype FPR2. Despite acting as a calcium-mobilizing agonist at FPR, Spinorphin was a weak chemotactic agonist and effectively blocked neutrophil chemotaxis induced by fMLF at concentrations selective for FPR. Spinorphin did not affect mouse neutrophil chemotaxis induced by concentrations of fMLF that selectively activate FPR2. Thus, Spinorphin blocks fMLF-induced neutrophil chemotaxis by acting as a specific antagonist at the fMLF receptor subtype FPR.