ABSTRACT
The physiological role of the distinct splice variants for cloned mouse µ-opioid receptor (mMOR-1), mMOR-1J, mMOR-1K and mMOR-1L, which are sensitive to N(α)-amidino-Tyr-D-Arg-Phe-ß-Ala (amidino-TAPA) and insensitive to [D-Ala(2),N-MePhe(4),Gly-ol(5)]enkephalin (DAMGO), was described in the mouse spinal cord. The antinociception induced by intrathecally (i.t.) injected amidino-TAPA was attenuated by i.t. pretreatment with antisera against the endogenous opioid peptides dynorphin A, dynorphin B, α-neo-endorphin, or [Leu(5)]enkephalin in naïve mice. However, in mice whose spinal mMOR-1J had been knocked-down using the antisense oligodeoxynucleotide (ODN) for exon-12 of mMOR-1 gene, the inhibiting effect of antiserum against dynorphin A on amidino-TAPA-induced antinociception was eliminated. In contrast, the inhibiting effect of antisera against dynorphin B or α-neo-endorphin on amidino-TAPA-induced antinociception was eliminated in mice whose spinal mMOR-1K had been knocked-down using the antisense ODN for exon-13 of mMOR-1 gene. Moreover, the inhibiting effect of antisera against dynorphin A or [Leu(5)]enkephalin on amidino-TAPA-induced antinociception was eliminated in mice whose spinal mMOR-1L had been knocked-down using the antisense ODN for exon-14 of mMOR-1 gene. The present results suggest that the distinct antinociceptive profile of amidino-TAPA, that is the release of endogenous κ- and δ-opioid peptides in spinal cord, is mediated through the activation of mMOR-1J, mMOR-1K or mMOR-1L, which contain the sequence encoded by exon-12, exon-13 or exon-14 of mMOR-1 gene, respectively.
Subject(s)
Analgesics/pharmacology , Enkephalin, Ala(2)-MePhe(4)-Gly(5)-/pharmacology , Oligopeptides/pharmacology , Protein Isoforms/metabolism , Receptors, Opioid, mu/metabolism , Spinal Cord/metabolism , Animals , Gene Knockdown Techniques , Male , Mice , Protein Isoforms/deficiency , Protein Isoforms/genetics , Receptors, Opioid, mu/deficiency , Receptors, Opioid, mu/genetics , Spinal Cord/drug effectsABSTRACT
The involvement of the mouse µ-opioid receptor (mMOR-1) splice variants in the antinociceptive effect of intrathecally (i.t.) administered N(α)-amidino-Tyr-D-Arg-Phe-ß-Ala (amidino-TAPA) and [D-Ala(2),N-MePhe(4),Gly-ol(5)]enkephalin (DAMGO) was investigated in mice by monitoring the recovery from acute antinociceptive tolerance to amidino-TAPA and DAMGO. A single i.t. pretreatment with DAMGO produced an acute antinociceptive tolerance, which peaked at 2h and disappeared within 5h after the pretreatment. In contrast, a single i.t. pretreatment with amidino-TAPA produced an acute antinociceptive tolerance, which disappeared within 3h after the pretreatment. The concomitant i.t. pretreatment with an antisense oligodeoxynucleotide (ODN) for exon-1, exon-12, exon-13 or exon-14 of mMOR-1 maintained the acute antinociceptive tolerance to amidino-TAPA for 24h after the pretreatment. On the other hand, the concomitant i.t. pretreatment with an antisense ODN for exon-1 of mMOR-1, but not an antisense ODN for exon-12, exon-13 or exon-14 of mMOR-1, maintained the acute antinociceptive tolerance to DAMGO for 24h after the pretreatment. The present results suggest that the spinal antinociception of amidino-TAPA is partially mediated through the activation of the amidino-TAPA-sensitive and DAMGO-insensitive mMOR-1 splice variants MOR-1J, MOR-1K and MOR-1L, which contain the sequence encoded by exon-12, exon-13 and exon-14, respectively.
Subject(s)
Analgesics/pharmacology , Oligopeptides/chemistry , Oligopeptides/pharmacology , Opioid Peptides/chemistry , Receptors, Opioid, mu/metabolism , Spinal Cord/drug effects , Spinal Cord/metabolism , Animals , Base Sequence , Enkephalin, Ala(2)-MePhe(4)-Gly(5)-/pharmacology , Exons/genetics , Male , Mice , Oligodeoxyribonucleotides, Antisense/genetics , Protein Isoforms/genetics , Protein Isoforms/metabolism , Receptors, Opioid, mu/geneticsABSTRACT
The antinociceptive effect of i.t. administered N(alpha)-amidino-Tyr-d-Arg-Phe-beta-Ala (amidino-TAPA), an N-terminal tetrapeptide analog of dermorphin, was characterized in ddY mice. In the opioid receptor ligand-binding assays using mouse brain membranes, amidino-TAPA showed a very high affinity for mu-opioid receptors, a low affinity to delta-opioid receptors and no affinity for kappa-opioid receptors. In the mouse tail-flick test, i.t. treatment with amidino-TAPA produced a potent antinociception. The antinociception induced by amidino-TAPA was significantly attenuated by i.t. pretreatment with the mu-opioid receptor antagonist beta-funaltrexamine, the kappa-opioid receptor antagonist nor-binaltorphimine and the delta-opioid receptor antagonist naltrindole. Moreover, the antinociception induced by amidino-TAPA was significantly attenuated by i.t. pretreatment with antisera against the endogenous kappa-opioid peptides dynorphin A, dynorphin B and alpha-neo-endorphin; and the endogenous delta-opioid peptide [Leu(5)]enkephalin. In mice lacking prodynorphin, the precursor of the endogenous kappa-opioid peptides, the antinociceptive effect of amidino-TAPA was significantly attenuated compared to that in wild-type C57BL/6J mice. However, there was no difference in G-protein activation by amidino-TAPA in the spinal cord membranes from prodynorphin knockout mice and C57BL/6J mice. The present results suggest that the spinal antinociception induced by the mu-opioid receptor selective peptide amidino-TAPA is mediated in part by the release of endogenous opioid peptides in the spinal cord, which is caused by the direct stimulation of mu-opioid receptors.
