Endomorphins 1 and 2 reduce relaxant non-adrenergic,non-cholinergic neurotransmission in rat gastric fundus

It is now well established that opioids modulate cholinergic excitatory neurotransmission in the gastrointestinal tract. The aim of the present study was to characterize a possible effect of endomorphins on nonadrenergic, noncholinergic (NANC) relaxant neurotransmission in the rat gastric fundus in vitro. The drugs used in the experiments were the endogenous mu-opioid receptors (MORs) endomorphin 1 and 2 and the mu-opioid receptor antagonist CTAP (D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2). CTAP left the basal tonus and the spontaneous activity of the preparation unchanged. Electrical field stimulation (EFS) under NANC conditions at frequencies ranging from 0.5 to 16 Hz caused a frequency-dependent relaxant response on the 5-hydoxytryptamine (5-HT) (10(-7) M) precontracted smooth-muscle strip. Both endomorphin 1 and endomorphin 2 significantly reduced this relaxation in a concentration-dependent manner. Endomorphin 1 proved to be more potent in reducing the relaxant responses. The endomorphin effects were significantly reversed by the MOR antagonist CTAP. CTAP itself did not influence the EFS-induced relaxation. In summary, these data provide evidence that the endogenous MOR agonists endomorphin 1 and 2 can reduce nonadrenergic, noncholinergic neurotransmission in the rat gastric fundus smooth muscle via a pathway involving MORs. The physiological relevance of these findings remains to be established, since the data presented suggest that the endomorphins act as neuromodulators within NANC relaxant neurotransmission.     

Endomorphin-Stimulated [35S]GTPγS Binding in Rat Brain: Evidence for Partial Agonist Activity at μ-Opioid Receptors

Abstract: Endomorphin-1 is a peptide whose binding selectivity suggests a role as an endogenous ligand at μ-opioid receptors. In the present study, the effect of endomorphin-1 on μ receptor-coupled G proteins was compared with that of the μ agonist DAMGO by using agonist-stimulated [³⁵S]GTPγS binding in rat brain. [³⁵S]GTPγS autoradiography revealed a similar localization of endomorphin-1 and DAMGO-stimulated [³⁵S]GTPγS binding in areas including thalamus, caudate-putamen, amygdala, periaqueductal gray, parabrachial nucleus, and nucleus tractus solitarius. Naloxone blocked endomorphin-1-stimulated labeling in all regions examined. Although the distribution of endomorphin-1-stimulated [³⁵S]GTPγS binding resembled that of DAMGO, the magnitude of endomorphin-1-stimulated binding was significantly lower than that produced by DAMGO. Concentration-effect curves of endomorphin-1 and DAMGO in thalamic membranes confirmed that endomorphin-1 produced only 70% of DAMGO-stimulated [³⁵S]GTPγS binding. Differences in maximal stimulation of [³⁵S]GTPγS binding between DAMGO and endomorphin-1 were magnified by increasing GDP concentrations, and saturation analysis of net endomorphin-1-stimulated [³⁵S]GTPγS binding revealed a lower apparent B _max value than that obtained with DAMGO. Endomorphin-1 also partially antagonized DAMGO stimulation of [³⁵S]GTPγS binding. These results demonstrate that endomorphin-1 is a partial agonist for G protein activation at the μ-opioid receptor in brain.     

The Effects of Endomorphins on Striatal [<Superscript>3</Superscript>H]Gaba Release Induced by Electrical Stimulation: An In vitro Superfusion Study in Rats

The endomorphins (EM1 and EM2) are selective endogenous ligands for mu-opioid receptors (MOR1 and MOR2) with neurotransmitter and neuromodulator roles in mammals. In the present study we investigated the potential actions of EMs on striatal GABA release and the implication of different MORs in these processes. Rat striatal slices were preincubated with tritium-labelled GABA ([³H]GABA), pretreated with selective MOR1 and MOR2 antagonist beta-funaltrexamine and selective MOR1 antagonist naloxonazine and then superfused with the selective MOR agonists, EM1 and EM2. EM1 significantly decreased the striatal [³H]GABA release induced by electrical stimulation. Beta-funaltrexamine antagonized the inhibitory action of EM1, but naloxonazine did not affect it considerably. EM2 was ineffective, even in case of specific enzyme inhibitor diprotin A pretreatment. The results demonstrate that EM1 decreases GABA release in the basal ganglia through MOR2, while EM2 does not influence it.     

Molecular modeling studies to predict the possible binding modes of endomorphin analogs in μ opioid receptor

The molecular docking of a series of endomorphin analog with the μ opioid receptor was performed. The successive molecular dynamics of several proposed ligand–receptor complexes inserted into the phospholipid bilayer were carried out to optimize the complex and explore the conformational changes. Meaningful differences of their binding modes were detected and the involvement of some essential residues in ligand binding was also identified. Our proposed ligand–receptor model is in good agreement with previous site-directed mutagenesis experiments.     

Design,synthesis, and biological activity of new endomorphin analogs with multi-site modifications

Endomorphin (EM)-1 and EM-2 are the most effective endogenous analgesics with efficient separation of analgesia from the risk of adverse effects. Poor metabolic stability and ineffective analgesia after peripheral administration were detrimental for the use of EMs as novel clinical analgesics. Therefore, here, we aimed to establish new EM analogs via introducing different bifunctional d-amino acids at position 2 of [(2-furyl)Map⁴]EMs. The combination of [(2-furyl)Map⁴]EMs with D-Arg² or D-Cit² yielded analogs with enhanced binding affinity to the μ-opioid receptor (MOR) and increased stability against enzymatic degradation (t_1/2 > 300 min). However, the agonistic activities of these analogs toward MOR were slightly reduced. Similar to morphine, peripheral administration of the analog [D-Cit², (2-furyl)Map⁴]EM-1 (10) significantly inhibited the pain behavior of mice in multiple pain models. In addition, this EM-1 analog was associated with reduced tolerance, less effect on gastrointestinal mobility, and no significant motor impairment. Compared to natural EMs, the EM analogs synthesized herein had enhanced metabolic stability, bioavailability, and analgesic properties.     

Actions of endomorphins on synaptic transmission of aδ-fibers in spinal cord dorsal horn neurons

The effects of endogenous mu-opioid ligands, endomorphins, on Adelta-afferent-evoked excitatory postsynaptic currents (EPSCs) were studied in substantia gelatinosa neurons in spinal cord slices. Under voltage-clamp conditions, endomorphins blocked the evoked EPSCs in a dose-dependent manner. To determine if the block resulted from changes in transmitter release from glutamatergic synaptic terminals, the opioid actions on miniature excitatory postsynaptic currents (mEPSCs) were examined. Endomorphins (1 microM) reduced the frequency but not the amplitude of mEPSCs, suggesting that endomorphins directly act on presynaptic terminals. The effects of endomorphins on the unitary (quantal) properties of the evoked EPSCs were also studied. Endomorphins reduced unitary content without significantly changing unitary amplitude. These results suggest that in addition to presynaptic actions on interneurons, endomorphins also inhibit evoked EPSCs by reducing transmitter release from Adelta-afferent terminals.     

From MIF-1 to endomorphin: the Tyr-MIF-1 family of peptides

The Tyr-MIF-1 family of small peptides has served a prototypic role in the introduction of several novel concepts into the peptide field of research. MIF-1 (Pro-Leu-Gly-NH₂) was the first hypothalamic peptide shown to act “up” on the brain, not just “down” on the pituitary. In several situations, including clinical depression, MIF-1 exhibits an inverted U-shaped dose–response relationship in which increasing doses can result in decreasing effects. This tripeptide also can antagonize opiate actions, and the first report of such activity also correctly predicted the discovery of other endogenous antiopiate peptides. The tetrapeptide Tyr-MIF-1 (Tyr-Pro-Leu-Gly-NH₂) not only shows antiopiate activity, but also considerable selectivity for the mu-opiate binding site. Tyr-W-MIF-1 (Tyr-Pro-Trp-Gly-NH₂) is an even more selective ligand for the mu receptor, leading to the discovery of two more Tyr-Pro tetrapeptides that have the highest specificity and affinity for this site. These are the endomorphins: endomorphin-1 is Tyr-Pro-Trp-Phe-NH₂ and endomorphin-2 is Tyr-Pro-Phe-Phe-NH₂. Tyr-MIF-1 proved, contrary to the then prevailing dogma, that peptides can be saturably transported across the blood–brain barrier by a quantifiable transport system. Unexpectedly, the Tyr-MIF-1 transporter is shared with Met-enkephalin. In the era in which it was doubtful whether a peripheral peptide could exert CNS effects, the Tyr-MIF-1 family of peptides also explicitly showed that they can exert more than one central action that persists longer than their half-lives in blood. These peptides clearly illustrate that the name of a peptide restricts neither its actions nor its conceptual implications.     

Synthesis, radiolabeling and receptor binding of [3H][(1S,2R)ACPC2]endomorphin-2

Previously, we have shown that substitution of Pro² for cis-2-aminocyclopentanecarboxylic acid, ACPC in endomorphin-2 results in an analogue with greatly augmented proteolytic stability, high μ-opioid receptor affinity and selectivity. We now report the synthesis and biochemical characterization of [³H][(1S,2R)ACPC²]endomorphin-2 with a specific activity of 1.41 TBq/mmol (38.17 Ci/mmol). Specific binding of [³H][(1S,2R)ACPC²]endomorphin-2 was saturable and of high affinity with an equilibrium dissociation constant, K_d = 1.80 ± 0.21 nM and receptor density, B_max = 345 ± 27 fmol × mg protein⁻¹ at 25 °C in rat brain membranes. Similar affinity values were obtained in kinetic and displacement assays. Both Na⁺ and Gpp(NH)p decreased the affinity proving the agonist character of the radioligand. [³H][(1S,2R)ACPC²]endomorphin-2 retained the μ-specificity of the parent peptide. The new radioligand will be a useful tool to map the topographical requirements of μ-opioid peptide binding due to its high affinity, selectivity and enzymatic stability.     

The structure of an endomorphin analogue incorporating 1-aminocyclohexane-1-carboxlylic acid for proline is similar to the beta-turn of Leu-enkephalin

Endomorphin (EM2, Tyr-Pro-Phe-Phe-NH(2)) can assume various conformations related to cis/trans-rotamers of the amide linkage of Tyr-Pro. To control isomerization, restricted or flexible components have been introduced at the Pro position. We focused on [Chx(2)]EM2, an EM2 analogue substituting 1-aminocyclohexane-1-carboxlylic acid (Chx) for Pro. X-ray diffraction analysis revealed that [Chx(2)]EM2 is folded into the trans-form of Tyr-Chx. The manner of folding resembled that seen in D-TIPP, an EM analogue incorporating tetrahydroisoquinoline carboxylic acid, as well as the beta-turn of Leu-enkephalin. Selectivity for the opioid mu-receptor was fairly well conserved by [Chx(2)]EM, suggesting that the folded form is important for mu-selectivity.     

Pharmacology of a new tritiated endomorphin-2 analog containing the proline mimetic cis-2-aminocyclohexanecarboxylic acid

As part of ongoing work aimed at generating proteolytically stable, readily applicable, radiolabeled endomorphin-2 (EM-2) analogs for elucidation of the topological requirements of peptide binding to μ-opioid receptors, we report here on the synthesis, radiolabeling, binding kinetics and binding site distribution of an EM-2 analog in which Pro² is replaced by 2-aminocyclohexanecarboxylic acid, ACHC. [³H][(1S,2R)ACHC]²EM-2 (specific activity 63.49 Ci × mmol⁻¹) bound specifically to its binding sites with high affinity (K_D = 0.55 ± 0.06 nM) and saturably, yielding a receptor density, B_max of 151 ± 4 fmol × mg protein⁻¹ in rat brain membranes. A similar affinity value was obtained in kinetic assays. Both Na⁺ and Gpp(NH)p decreased the affinity, proving the agonist character of the radioligand. Specific μ-opioid ligands displaced the radioligand with much higher affinities than did δ- and κ-ligands. The autoradiographic distribution of the binding sites of [³H][(1S,2R)ACHC]²EM-2 agreed well with the known locations of the μ-opioid receptors in the rat brain. In consequence of its high affinity, selectivity and enzymatic resistance [19], the new radioligand will be a good tool in studies of the topographical requirements of μ-opioid-specific peptide binding.