[Met5]-Enkephalin, amide (5-Methionine-enkephalin amide)

Substrate- and inhibitor-specificity of a non-endothelial enzyme which forms [Met5]-enkephalin from [Met5]-enkephalin-Arg6,Phe7 in isolated rabbit ear artery: pharmacological characterization

The captopril-inhibited enzyme which forms [Met5]-enkephalin from [Met5]-enkephalin-Arg6,Phe7 in isolated rabbit ear artery was characterized further by using various natural substrate candidates/analogues ([Met5]-enkephalin-Arg6,Phe7 and its amide, [Met5]-enkephalin, angiotensin I and bradykinin), peptidase inhibitors such as captopril, enalaprilate and thiorphan and by endothelial removal. 10(-5) and 10(-4) M but not 10(-6) M captopril reduced the effectiveness of [Met5]-enkephalin-Arg6,Phe7 and potentiated the effect of bradykinin but did not affect markedly the action of the other peptides. Of the inhibitors, enalaprilate was less effective than captopril, and thiorphan had no effect. The [Met5]-enkephalin-Arg6,Phe7-->[Met5]-enkephalin conversion was not affected by endothelial removal. The substrate and inhibitor spectrum of this non-endothelial enzyme activity bears no relationship in other, hitherto characterized dipeptidylcarboxypeptidases/endopeptidases known to be involved in the metabolism of the tested peptides.

Acute effects of D-1 and D-2 dopamine receptor agonist and antagonist drugs on basal ganglia [Met5]- and [Leu5]-enkephalin and neurotensin content in the rat

The effects of acute systemic injection of the D-1 agonist SKF 38393 (2.5-20 mg/kg) or the D-1 antagonist SCH 23390 (0.25-2.0 mg/kg), and of the D-2 agonist quinpirole (0.12-1.0 mg/kg) or the D-2 antagonist sulpiride (25-100 mg/kg) on the neuropeptide content of rat basal ganglia were investigated. In striatum, the [Met5]- and [Leu5]-enkephalin content was unaffected by administration of SKF 38393 or SCH 23390. Quinpirole had no effect on [Met5]- and [Leu5]-enkephalin levels but sulpiride produced an increase in both [Met5]- and [Leu5]-enkephalin content. In the nucleus accumbens, SKF 38393 decreased and SCH 23390 increased [Met5]- and [Leu5]enkephalin levels. Quinpirole decreased [Met5]- and [Leu5]-enkephalin levels, while sulpiride decreased [Leu5]-enkephalin levels alone. The content of [Leu5]- but not [Met5]-enkephalin levels in the substantia nigra was increased by administration of SKF 38393, and decreased by SCH 23390. Quinpirole and sulpiride were without effect on the [Met5]- or [Leu5]-enkephalin content of substantia nigra. Neurotensin levels in striatum were increased by administration of SKF 38393 and decreased by SCH 23390. Similarly, quinpirole decreased the neurotensin content while sulpiride caused an increase. In the nucleus accumbens, the neurotensin content was not affected by administration of SKF 38393 but increased by SCH 23390. Neither quinpirole nor sulpiride altered neurotensin levels in the nucleus accumbens. Neurotensin levels in substantia nigra were unaffected by the administration of SKF 38393 and SCH 23390, or by quinpirole and sulpiride. These results indicate that acute administration of D-1 and D-2 agonist and antagonist drugs can alter the levels of [Met5]- and [Leu5]-enkephalin and neurotensin in basal ganglia. However, there are marked differences between brain regions in the regulation of peptide levels by acute D-1 and D-2 receptor occupation.

Glial growth is regulated by agonists selective for multiple opioid receptor types in vitro

To determine whether one or more opioid receptor types might be preferentially involved in gliogenesis, primary mixed glial cultures derived from mouse cerebra were continuously treated with varying concentrations of opioid agonists selective for mu (mu), i.e., DAGO ([D-Ala2, MePhe4, Gly(ol)5]enkephalin), delta (delta), i.e., DPDPE ([D-PEN2,D-PEN5]enkephalin), or kappa (kappa), i.e., U69,593, opioid receptor types. In addition, a group of cultures was treated with [Met5]-enkephalin, an agonist for delta opioid receptors as well as putative zeta (zeta) opioid receptors. Opioid-dependent changes in growth were assessed by examining alterations in (1) the number of cells in mixed glial cultures at 3, 6, and 8 days in vitro (DIV), (2) [3H]thymidine incorporation by glial fibrillary acidic protein (GFAP) immunoreactive, flat (type 1) astrocytes at 6 DIV, and (3) the area and form factor of GFAP-immunoreactive, flat (type 1) astrocytes. DPDPE at 10(-8) or 10(-10) M, as well as [Met5]-enkephalin at 10(-6), 10(-8), or 10(-10) M, significantly reduced the total number of glial cells in culture; but this effect was not observed with DAGO or U69,593 (both at 10(-6), 10(-8), or 10(-10) M). Equimolar concentrations (i.e., 10(-6) M) of [Met5]enkephalin or U69,593, but not DPDPE or DAGO, suppressed the rate of [3H]thymidine incorporation by GFAP-immunoreactive, flat (type 1) astrocytes. DAGO had no effect on growth, although in previous studies morphine was found to inhibit glial numbers and astrocyte DNA synthesis. [Met5]enkephalin (10(-6) M) was the only agonist to significantly influence astrocyte area. Collectively, these results indicate that delta (and perhaps mu) opioid receptor agonists reduce the total number of cells in mixed glial cultures; while [Met5]enkephalin-responsive (and perhaps kappa-responsive) opioid receptors mediate DNA synthesis in astrocytes. This implies that delta opioid receptors, as well as [Met5]enkephalin-sensitive, non-delta opioid receptors, mediate opioid-dependent regulation of astrocyte and astrocyte progenitor growth. These data support the concept that opioid-dependent changes in central nervous system growth are the result of endogenous opioid peptides acting through multiple opioid receptor types.

Met5-enkephalin-Arg6-Phe7, an endogenous neuropeptide, binds to multiple opioid and nonopioid sites in rat brain

Receptor binding properties of the naturally occurring opioid heptapeptide MERF were studied in rat brain membrane preparations using tritium-labeled derivative of the peptide with 40 Ci/mmol specific radioactivity. Binding assays were performed in the presence of broad-spectrum peptidase inhibitors at 0 degree C. Under these conditions, the equilibrium binding was achieved in 30-40 min, and approximately 90% of the applied radioligand remained unchanged as determined by HPLC analysis. The apparent affinity (Kd value) of [3H]Met-enkephalin-Arg6-Phe7, calculated from saturation binding data, was 10.2 +/- 2.5 nM, and the maximal number (Bmax) of the heptapeptide binding sites was found to be 468 +/- 43 fmol/mg protein. About half the sites represent nonopioid sites because the Bmax was only 255 +/- 30 fmol/mg, when the nonspecific binding was measured with 1 microM naloxone. The rank order potencies of the examined compounds revealed that the opioid component of [3H]Met-enkephalin-Arg6-Phe7 recognition site are probably not mu and certainly not kappa 1 sites, whereas these sites are characterized by a kappa 2-like binding profile. Considering the discrepancies between rat and frog brain found in the affinity of some compounds, including naltrindole and norbinaltorphimine, the presence of a novel, MERF-selective "heptapeptide" binding site in rat brain membranes is also suggested. A number of the heterologous competition curves could be described by a high-affinity stereospecific component and a substantially lower-affinity binding element, which could completely be displaced with several peptide ligands such as Met5-enkephalin, dynorphin(1-13), and unlabeled MERF but not by other compounds such as [D-Ala2-(Me)Phe4-Gly5-ol]enkephalin, morphine, or naloxone. [3H]Met-enkephalin-Arg6-Phe7 binding can also be inhibited by FMRF-amide analogs and sigma receptor ligands, such as (+)N-allyl-normetazocine and haloperidol, although with moderate affinity. It is concluded that the stereospecific high-affinity binding is of opioid in character, whereas the residual sites characterized with their lower affinity are naloxone-insensitive nonopioid sites.

Activation of delta- and kappa-opioid receptors by opioid peptides protects cardiomyocytes via KATP channels

To examine the receptor specificity and the mechanism of opioid peptide-induced protection, we examined freshly isolated adult rabbit cardiomyocytes subjected to simulated ischemia. Cell death as a function of time was assessed by trypan blue permeability. Dynorphin B (DynB) and Met5-enkephalin (ME) limitation of cell death (expressed as area under the curve) was sensitive to blockade by naltrindole (NTI, a delta-selective antagonist) and 5'-guanidinyl-17-(cyclopropylmethyl)-6,7-dehydro-4,5alpha-epoxy-3,14-dihydroxy-6,7-2',3'-indolomorphinan (GNTI dihydrochloride, a kappa-selective antagonist): 85.7 +/- 2.7 and 142.9 +/- 2.7 with DynB and DynB + NTI, respectively (P < 0.001), 94.1 +/- 4.2 and 164.5 +/- 7.3 with DynB and DynB + GNTI, respectively (P < 0.001), 111.9 +/- 7.0 and 192.1 +/- 6.4 with ME and ME + NTI, respectively (P < 0.001), and 120.2 +/- 4.3 and 170.0 +/- 3.3 with ME and ME + GNTI, respectively (P < 0.001). Blockade of ATP-sensitive K+ channels eliminated DynB- and ME-induced protection: 189.6 +/- 5.4 and 139.0 +/- 5.4 for control and ME, respectively (P < 0.001), and 210 +/- 5.9 and 195 +/- 6.1 for 5-HD and ME + 5-HD, respectively (P < 0.001); 136.0 +/- 5.7 and 63.4 +/- 5.4 for control and ME, respectively (P < 0.001), and 144.6 +/- 4.5 and 114.6 +/- 7.7 for HMR-1098 and ME + HMR-1098, respectively (P < 0.01); 189.6 +/- 5.4 and 139.0 +/- 5.4 for control and ME, respectively (P < 0.001), and 210 +/- 5.9 and 195 +/- 6.1 for 5-HD and ME + 5-HD, respectively (P < 0.001); and 136.0 +/- 5.7 and 63.4 +/- 5.4 for control and ME, respectively (P < 0.001), and 144.6 +/- 4.5 and 114.6 +/- 7.7 for HMR-1098 and ME + HMR-1098, respectively (P < 0.01). We conclude that opioid peptide-induced cardioprotection is mediated by delta- and kappa-receptors and involves sarcolemmal and mitochondrial ATP-sensitive K+ channels.