In vivo regulation of the μ opioid receptor: role of the endogenous opioid agents

Abstract

It is well known that genotypic differences can account for the subject-specific responses to opiate administration. In this regard, the basal activity of the endogenous system (either at the receptor or ligand level) can modulate the effects of exogenous agonists as morphine and vice versa. The μ opioid receptor from zebrafish, dre-oprm1, binds endogenous peptides and morphine with similar affinities. Morphine administration during development altered the expression of the endogenous opioid propeptides proenkephalins and proopiomelanocortin. Treatment with opioid peptides (Met-enkephalin [Met-ENK], Met-enkephalin-Gly-Tyr [MEGY] and β-endorphin [β-END]) modulated dre-oprm1 expression during development. Knocking down the dre-oprm1 gene significantly modified the mRNA expression of the penk and pomc genes, thus indicating that oprm1 is involved in shaping penk and pomc expression. In addition, the absence of a functional oprm1 clearly disrupted the embryonic development, since proliferation was disorganized in the central nervous system of oprm1-morphant embryos: mitotic cells were found widespread through the optic tectum and were not restricted to the proliferative areas of the mid- and hindbrain. Transferase-mediated dUTP nick-end labeling (TUNEL) staining revealed that the number of apoptotic cells in the central nervous system (CNS) of morphants was clearly increased at 24-h postfertilization. These findings clarify the role of the endogenous opioid system in CNS development. Our results will also help unravel the complex feedback loops that modulate opioid activity and that may be involved in establishing a coordinated expression of both receptors and endogenous ligands. Further knowledge of the complex interactions between the opioid system and analgesic drugs will provide insights that may be relevant for analgesic therapy.

Figure 1

Pharmacological properties of endogenous opioid peptides on dre-μ. Competition binding curves of endogenous opioid peptides on dre-μ membrane homogenates are shown. Data were fit to the one-site competition model, and each point represents the mean ± standard error of the mean (SEM) (capped bars) of three independent experiments performed in triplicate. Morphine, a μ agonist, and naloxone, a non-specific antagonist, were added for comparison. □, Met-ENK; △, Leu-ENK; ▽, β-END; ○, Met-ENK-Gly-Tyr (MEGY); +, (d-Ala²)-MEGY; ×, (d -Ala², Val⁵)-MEGY; *, morphine; ■, naloxone.

Figure 2

Effect of opioid peptide treatment of the developmental pattern of dre-oprm1. dre-oprm1 expression was assayed by in situ hybridization on whole-mount embryos that were exposed to 10 μmol/L Met-ENK, MEGY or β-END from 5 to 24 hpf or 48 hpf. Embryos are oriented anterior toward the left and posterior toward the right. A total of 25 embryos were used for each treatment and for each time point, and the experiment was repeated three times.

Figure 3

Morphine effect on the expression of the opioid propeptides. Zebrafish embryos were exposed to 10 nmol/L morphine, and penk a, penk b and pomc a expression was established at 24 and 48 hpf and compared with controls. Embryos are oriented anterior toward the left and posterior toward the right. A total of 25 embryos were used for each treatment and for each time point, and the experiment was repeated three times.

Figure 4

Effect of knocking down dre-oprm1 on the expression pattern of the opioid precursors. The expression of proenkephalins (dre-penk a and dre-penk b) and proopiomelanocortin (dre-pomc a) was assayed in dre-oprm1 morphants of 24 and 48 hpf. Embryos are oriented anterior toward the left and posterior toward the right. Arrowheads indicate the border between tegmentum and optic tectum; and arrows indicate the most posterior area of the medulla oblongata. A total of 25 embryos were used for each treatment and for each time point, and the experiment was repeated three times. mor MO, morpholino designed against dre-oprm1 gene.

Figure 5

Proliferation studies on dre-oprm1 morphants. Phospho-histone 3 (Ser¹⁰) immunostaining (red) was assayed on whole-mounts of control and dre-oprm1 morphants of 24 and 48 hpf. The cell nuclei are counterstained with DAPI (blue) to give a better overview of the embryo. Dorsal views of the flat-mounts are shown, and embryos are oriented anterior toward the left and posterior toward the right. A total of 25 embryos were used for each treatment and for each time point, and the experiment was repeated three times. dre-mor MOR, morpholino designed against dre-oprm1 gene.

Figure 6

Apoptosis assays on dre-oprm1 morphants. TUNEL staining on control and dre-oprm1 morphants of 24 and 48 hpf. The apoptotic nuclei are positive for peroxidase labeling. Lateral views are shown, and embryos are oriented anterior toward the left and posterior toward the right. A total of 25 embryos were used for each treatment and for each time point, and the experiment was repeated three times.