tyrosyl-1-2-3-4-tetrahydro-3-isoquinolinecarbonyl-phenylalanyl-phenylalanine and deltorphin

The pontine parabrachial nucleus (PBN) receives both opioid and Neuropeptide FF (NPFF) projections from the lower brain stem and/or the spinal cord. Because of this anatomical convergence and previous evidence that NPFF displays both pro- and anti-opioid activities, this study examined the synaptic effects of NPFF in the PBN and the mechanisms underlying these effects using an in vitro brain slice preparation and the nystatin-perforated patch-clamp recording technique. Under voltage-clamp conditions, NPFF reversibly reduced the evoked excitatory postsynaptic currents (EPSCs) in a dose-dependent fashion. This effect was not accompanied by apparent changes in the holding current, the current-voltage relationship or alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-induced inward currents in the PBN cells. When a paired-pulse protocol was used, NPFF increased the ratio of these synaptic currents. Analysis of miniature EPSCs showed that NPFF caused a rightward shift in the frequency-distribution curve, whereas the amplitude-distribution curve remained unchanged. Collectively, these experiments indicate that NPFF reduces the evoked EPSCs through a presynaptic mechanism of action. The synaptic effects induced by NPFF (5 microM) could not be blocked by the specific mu-opioid receptor antagonist, D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH(2) (1 microM), but application of delta-opioid receptor antagonist Tyr-Tic-Phe-Phe (5 microM) almost completely prevented effects of NPFF. Moreover, the delta-opioid receptor agonist, Deltorphin (1 microM), mimicked the effects as NPFF and also occluded NPFF's actions on synaptic currents. These results indicate that NPFF modulates excitatory synaptic transmission in the PBN through an interaction with presynaptic delta-opioid receptors. These observations provide a cellular basis for NPFF enhancement of the antinociceptive effects consequent to central activation of delta-opioid receptors.

Topics: Analgesics, Opioid; Animals; Dose-Response Relationship, Drug; Electric Conductivity; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Excitatory Postsynaptic Potentials; GABA Antagonists; In Vitro Techniques; Ligands; Male; Naloxone; Narcotic Antagonists; Oligopeptides; Patch-Clamp Techniques; Peptides; Picrotoxin; Pons; Potassium; Presynaptic Terminals; Rats; Rats, Sprague-Dawley; Receptors, Opioid, delta; Receptors, Opioid, mu; Tetrahydroisoquinolines; Tetrodotoxin

The N-terminal dipeptide Tyr-d-Ala of a mu-selective agonist, dermorphin tetrapeptide (DT, H-Tyr-D-Ala-Phe-Gly-NH2) and delta-selective agonist deltorphin C (DEL-C, H-Tyr-D-Ala-Phe-Asp-Val-Val- Gly-NH2) was changed into an aminodiacyl moiety. The relevant synthetic step is a nucleophilic substitution of bromine from a chiral 2-bromopropanamide by the amino group of tyrosine, with overall retention of configuration. The resulting pseudo tetra- and heptapeptides I-VI were characterized for mu- and delta-opioid receptor binding properties using [3H]DAGO and [3H]DPDPE, respectively, and in a bioassay using guinea pig ileum (GPI) and mouse vas deferens (MVD). As a result of chemical alteration of N-terminal depeptide moiety, all synthesized analogs showed considerable reduction in opioid receptor affinity compared to mu- and delta-prototypes (500-fold on the mu-site, analog I, and 125-fold on the delta-site, analog IV). Interestingly, analogs I and IV showed moderate antagonist activity, respectively, on GPI and MVD, with pA2 values of 6.05 and 6.82. Analog IV did not exhibit the delta-antagonist potency and delta-selectivity of TIPP peptides.

Topics: Amino Acid Sequence; Animals; Binding Sites; Binding, Competitive; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalins; Guinea Pigs; Ileum; Male; Mice; Molecular Sequence Data; Molecular Structure; Morphine; Narcotic Antagonists; Oligopeptides; Opioid Peptides; Receptors, Opioid; Stereoisomerism; Tetrahydroisoquinolines; Vas Deferens