Characterization of rat spinal cord receptors to FLFQPQRFamide, a mammalian morphine modulating peptide: a binding study

doi:10.1016/0006-8993(89)90311-9

Brain Research

Volume 500, Issues 1–2, 23 October 1989, Pages 169-176

https://doi.org/10.1016/0006-8993(89)90311-9 Get rights and content

Abstract

An in vitro binding assay, using ¹²⁵I-YLFQPQRFamide, a newly synthetized iodinated analog of FLFQPQRFamide, in which Phe¹ (F) has been substituted by a Tyr (Y), was developed to demonstrate and characterize putative binding sites of this brain morphine modulating peptide. This radioligand bound in a time-dependent manner to rat spinal cord membrane preparation. This binding was dose-dependent, saturable and reversible. Both kinetic data and saturation measured at equilibrium lead to the existence of a homogenous population of high affinity binding sites with a K_d value of 0.09–0.1 nM and a maximal capacity B_max of 14.5 ± 2fmol/mg protein. Results of competition experiments show that both FLFQPQRFamide and its analog YLFQPQRFamide had a similar capacity to inhibit the ¹²⁵I-YLFQPQRFamide binding, suggesting that this radioiodinated analog is a good tool to study binding characteristics of FLFQPQRFamide receptors. The related octadecapeptide AGEGLSSPFWSLAAPQRFamide, another mammalian morphine modulating peptide competes for radioligand binding with similar potency. Our results also show that μ, δ and κ opiate receptor agonists as well as the antagonist naloxone were not able to affect binding either in presence or in absence of 120 mM NaCl. Together, these data demonstrate that FLFQPQRFamide does not function as an endogenous opiate receptor antagonist and that its capacity to reduce opiate-induced analgesia is supported by specific binding sites.

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This article is part of the Special Issue entitled ‘Acid-Sensing Ion Channels in the Nervous System’.
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Possible in vivo interference between downstream signaling of MOP and NPFF-R2 has been found in some brain regions (nucleus accumbens, ventral pallidum) [88]. However, a selective NPFF-R2 agonist effectively counteracts with opioid-analgesia [32], suggesting that anti-opioid effect of NPFF may be also evoked independently from MOP or KOP [2], possibly through a direct NPFF-R2 and NPFF-R1 activation [70]. Interestingly, both acute morphine treatment [99] and ablation of opioid receptor genes [46] evoke region-dependent upregulation of NPFF-R2 expression in the murine brain.
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The physiological role of NPFF/FMRFa family of peptides appears to be complex and exact mechanism of action of these peptides is not yet completely understood. In same line of scrutiny, another analog of YGGFMKKKFMRFamide (YFa), a chimeric peptide of met-enkephalin and FMRFamide, was rationally designed and synthesized which contain D-alanine and p-Cl-phenylalanine residues at 2nd and 4th positions, respectively i.e., Y-(D-Ala)-G-(p-Cl-Phe)-MKKKFMRFamide ([D-Ala², p-Cl-Phe⁴]YFa) in order to achieve improved bioavailability and blood brain barrier penetration. Therefore, present study investigates the possible antinociceptive effect of [D-Ala², p-Cl-Phe⁴]YFa on intra-peritoneal (i.p.) administration using tail-flick test in rats followed by its opioid receptor(s) specificity using μ, δ and κ receptor antagonists. Further, its antinociceptive effect was examined during 6 days of chronic i.p. treatment and assessed effect of this treatment on differential expression of opioid receptors. [D-Ala², p-Cl-Phe⁴]YFa in comparison to parent peptide YFa, induce significantly higher dose dependent antinociception in rats which was mediated by all three opioid receptors (μ, δ and κ). Importantly, it induced comparable antinociception in rats throughout the chronic i.p. treatment and significantly up-regulated the overall expression (mRNA and protein) of μ, δ and κ opioid receptors. Therefore, pharmacological and molecular behavior of [D-Ala², p-Cl-Phe⁴]YFa demonstrate that incorporation of D-alanine and p-Cl-phenylalanine residues at appropriate positions in chimeric peptide leads to altered opioid receptor selectivity and enhanced antinociceptive potency, relative to parent peptide.

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Characterization of rat spinal cord receptors to FLFQPQRFamide, a mammalian morphine modulating peptide: a binding study

Abstract

Anal. Biochem.

Life Sci.

Brain Research

Biochem. Biophys. Res. Commun.

Peptides

Peptides

Peptides

J. Biol. Chem.

Complete structure of the porcine pro-opiomelanocortin mRNA derived from the nucleotide sequence of cloned cDNA

Nucl. Acids Res.

Modulation of potassium conductances by an endogenous neuropeptide in neurones ofAplysia california

J. Physiol. (Lond.)

The neuropeptide FMRFamide decreases both the Ca2+ conductance and a cyclic 3′,5′-adenosine monophosphate-dependent K+ conductance in identified molluscan neurons

J. Neurosci.

Bremazocine is an agonist at kappa opioid receptors and an antagonist at μ-opioid receptors in the guinea-pig myenteric plexus

Br. J. Pharmacol.

FMRF amide neuropeptides simultaneously increase and decrease K+ currents in an identified neurone

Nature (Lond.)

Antagonism of μ-opioid receptor-mediated inhibitions of nociceptive neurones by U50488H and dynorphin A(1–13) in the rat dorsal horn

Neurosci. Lett.

Mammalian neuronal actions of FMRFamide and the structurally related opioid Met-enkephalin-Arg6-Phe7

Nature (Lond.)

The neuropeptide FMRFamide decreases both the Ca²⁺ conductance and a cyclic 3′,5′-adenosine monophosphate-dependent K⁺ conductance in identified molluscan neurons

FMRF amide neuropeptides simultaneously increase and decrease K⁺ currents in an identified neurone

Mammalian neuronal actions of FMRFamide and the structurally related opioid Met-enkephalin-Arg⁶-Phe⁷