S. Salvadori

New features of the δ opioid receptor: Conformational properties of deltorphin I analogues

Deltorphin I is an opioid peptide of sequence H-Tyr-D-Ala-Phe-Asp-Val-Val-Gly-NH2, recently isolated from the skin of Phyllomedusa bicolor. Its enormous selectivity towards the delta opioid receptor and the similarity of the conformation of the N-terminal part of the sequence with that of dermorphin (H-Tyr-D-Ala-he-Gly-Tyr-Pro-Ser-NH2), a mu selective peptide, prompted the synthesis, biological evaluation and comparative conformational study of four analogs. A 1H-NMR study showed that the conformational preferences of the N-terminal sequences of all peptides are similar. The different selectivities towards opioid receptors have been interpreted in terms of charge effects in the interaction with the membrane and at the receptor site and of hydrophobicity of the C-terminal part, when structured in a folded conformation.

Na+ mechanism of δ-opioid receptor induced protection from anoxic K+ leakage in the cortex

Abstract.Activation of δ-opioid receptors (DOR) attenuates anoxic K+ leakage and protects cortical neurons from anoxic insults by inhibiting Na+ influx. It is unknown, however, which pathway(s) that mediates the Na+ influx is the target of DOR signal. In the present work, we found that, in the cortex, (1) DOR protection was largely dependent on the inhibition of anoxic Na+ influxes mediated by voltage-gated Na+ channels; (2) DOR activation inhibited Na+ influx mediated by ionotropic glutamate N-methyl-D-aspartate (NMDA) receptors, but not that by non-NMDA receptors, although both played a role in anoxic K+ derangement; and (3) DOR activation had little effect on Na+/Ca2+ exchanger-based response to anoxia. We conclude that DOR activation attenuates anoxic K+ derangement by restricting Na+ influx mediated by Na+ channels and NMDA receptors, and that non-NMDA receptors and Na+/Ca2+ exchangers, although involved in anoxic K+ derangement in certain degrees, are less likely the targets of DOR signal.

Conformational properties of deltorphin: new features of the δ-opioid receptor

Deltorphin is an opioid peptide with the sequence H‐Tyr‐D‐Met‐Phe‐His‐Leu‐Met‐Asp‐NH2, recently isolated from the skin of Phyllomedusa sauvagei. Its enormous selectivity towards the δ‐opioid receptor and the similarity of the N‐terminal part of the sequence with that of dermorphin (H‐Tyr‐D‐Ala‐Phe‐Gly‐Tyr‐Pro‐Ser‐NH2), a μ selective peptide isolated from the same natural source, prompted a comparative conformational study. A 1H‐NMR study in two different solvent systems showed that the conformational preferences of the N‐terminal sequences of the two peptides are similar. The different selectivities towards opioid receptors have been interpreted in terms of charge effects. Besides a general trend consistent with the role of the membrane in the preselection of the peptides, the present study demonstrates the crucial role played by charged residues in the interaction inside the receptors.

Design of μ selective opioid dipeptide antagonists

We have recently designed potent δ selective opioid antagonist dipeptides on the basis of a simple conformational analysis. Following a similar procedure we found a μ selective dipeptide antagonist, 2,6‐dimethyl‐Tyr‐d‐Phe‐NH2. Although its selectivity is not as high as those of the quoted δ selective dipeptides it has good in vitro activity and looks very promising for further development since the 2,6‐dimethyl‐Tyr‐D‐Phe message, like the δ selective 2,6‐dimethyl‐Tyr‐1,2,3,4‐tetrahydroisoquinoline‐3‐carboxylic acid counterpart, seems able to impart antagonism to longer peptides.

Reversed-phase HPLC study on the in vitro enzymic degradation of dermorphin

A high-performance liquid chromatographic (HPLC) method for the separation of the opioid heptapeptide dermorphin and related fragments has been developed. The chromatographic system was applied in the study of the kinetics of degradation of dermorphin (Der) in various tissues. Der was found to be extremely resistant to human and rat plasma (T 1/2 greater than 180 min). Upon incubation with homogenates of rat brains and kidneys, Der was cleaved with a half-life of 20.8 +/- 2.2 min and 2.4 +/- 0.3 min respectively. The catabolite formed was identified, in both tissues, as the N-terminal tetrapeptide H-Tyr-D-Ala-Phe-Gly-OH. The stability to rat kidney and brain of the N-terminal hexa- and pentapeptides and of the [4 psi 5, NHCO] Der analogue was also investigated. The nature of the enzyme systems involved in the in vitro degradations is discussed.

Involvement of μ-opioid receptors in the modulation of pituitary-adrenal axis in normal and stressed rats

The availability of the most selective, high-affinity, natural opioid agonists for μ-receptors (dermorphin-DM) and δ-receptors (deltorphin-DT) has provided the possibility for in vivo studying of the role of acute and chronic activation of μ-and δ-opioid receptors on the functional activity of the hypothalamus-pituitary-adrenocortical (HPA) axis, both in basal conditions and in response to an acute stress in adult male rats. Plasma corticosterone (CS) and ß-endorphin-like-immunoreactivity (ß-EP-LI) levels were measured by specific radioimmunoassays before and after 5 and 30 minutes from the exposure to cold (3±0.5 C) water and forcing them to swim for 10 minutes (acute cold swimming stress). Acute administration of DM, the specific μ-receptor agonist, enhanced basal and stress induced plasma levels of CS and ß-EP-LI. These effects were antagonized by pretreatment with nalox-one, specific μ-opioid receptor antagonist, but not by naltrindole, a δ-opioid receptor antagonist. Long-term administration of DM did not alter resting plasma levels of CS and ß-EP-LI, but significantly reduced stress-induced increase of these hormones. Both the acute and chronic administration of the DT, highly selective δ-opioid receptors agonist, failed to modify resting and stress induced hormone levels. Our present data show that DM throughout μ-opioid receptors, but not DT, modulates the response of HPA axis to acute stress in rats, increasing or decreasing the release of CS and ß-EP-LI when acutely or chronically administered, respectively.

Conformational analysis of potent and very selective delta opioid dipeptide antagonists

The δ selectivity and antagonism of peptides containing l‐tetrahydro‐3‐isoquinoline carboxylic acid (Tic) in second position can be attributed mainly to the Tyr‐Tic unit. These properties can be further enhanced by substituting Tyr1 with 2,6‐dimethyl‐l‐tyrosyl (Dmt). Dmt‐Tic‐NH2, Dmt‐Tic‐OH, Dmt‐Tic‐Ala‐NH2 and Dmt‐Tic‐Ala‐OH are all more active and/or selective than the corresponding [Tyr1]‐parent peptides. In fact the selectivities of Dmt‐Tic‐OH and Dmt‐Tic‐Ala‐OH are the highest ever recorded for opioid molecules. 1H NMR spectra in a DMSO/water mixture at 278 K reveal the presence of two similar conformers, characterised by a cis or trans Dmt‐Tic bond, in all four peptides. A detailed conformational analysis in solution of Dmt‐Tic‐NH2 shows that these conformers have a shape very similar to that of the bioactive conformation of Tyr‐Tic‐NH2 and to that of naltrindole.

Dermorphin decreases plasma LH levels in human: Evidence for a modulatory role of gonadal steroids

The purpose of this study was to evaluate the effects of dermorphin, a new synthetic powerful opiate-like heptapeptide, on plasma luteinizing hormone (LH) and follicle stimulating hormone (FSH) levels in fertile and postmenopausal women. In fertile subjects, dermorphin (5.5 micrograms/kg min for 30 min) decreases plasma LH (p less than 0.01 vs. baseline and placebo values), but not plasma FSH. The area under the curve during dermorphin infusion was significantly lower than during placebo infusion (p less than 0.01). Pretreatment with the opioid receptor antagonist naloxone, blocked the decrease of plasma LH levels. In postmenopausal women not subjected to any treatment, dermorphin infusion did not significantly modify plasma LH and FSH levels. On the contrary, its administration to postmenopausal subjects treated with conjugated estrogens and medroxyprogesterone acetate significantly decreased plasma LH levels (p less than 0.01, vs. baseline, placebo and area under the curve). Considering the modulatory role exerted by ovarian steroids on the activity of such receptors, these data also indicate that opioid systems play a very important part in the hypothalamus-pituitary-ovarian axis.

Unique sequence in deltorphin C confers structural requirement for δ opioid receptor selectivity

Abstract A series of deltorphin C (H-Tyr- d -Ala-Phe-Asp-Val-Val-Gly-NH 2 ) analogues were synthesized to assess the consequences of changing anionic and hydrophobic residues on δ receptor selectivity. Analogues with altered C-terminal groups, inverted sequences, or esterified with tert -butyl, benzyl, or ethyl groups revealed that high δ selectivity required an unmodified amino acid sequence. Shifts of Asp and hydrophobic residues decreased δ selectivity due to loss in δ affinity (5- to ≈ 700-fold); μ affinity was unchanged or increased 14-fold. Suppression of charge or deamidation diminished δ selectivity through reduced δ and modified μ affinities. Data provide evidence that a negative charge does not a priori guarantee high selectivity and specific alignment of anionic and hydrophobic residues might facilitate optimum spatial configuration which complements the δ receptor binding site.

Dermorphin, a new opioid peptide, stimulates thyrotropin secretion in normal subjects

The effect of a recently described, potent opioid peptide, dermorphin (DER), on TSH secretion in euthyroid subjects has been studied. DER infused at a rate of 5.5 μg/Kg/min for30 min induced a significant increase in serum TSH concentration at 60,90, and 120 min after the infusion was begun. Treatment with naloxone administered 30 min before the DER infusion with a bolus dose of 4 mg, followed by a constant infusion of 1 μ/Kg/min for 150 min, prevented the rise in serum TSH. Naloxone administered alone did not induce any change in TSH concentration. The present findings suggest that DER has a stimulatory effect on TSH secretion, probably mediated by opioid receptors. These results, however, do not solve the question as to whether opioids have a physiological role in the control of pituitary TSH secretion.