enkephalin--ala(2)-mephe(4)-gly(5)- and deltorphin

The goal of the present study was to establish the behavioral role of the nucleus accumbens (Nacc) core in the feed-forward spiraling striato-nigro-striatal circuitry that transmits information from the Nacc shell toward the dorsal subregion of the neostriatum (DS) in freely moving rats. Unilateral injection of μ-opioid receptor agonist [D-Ala(2),N-Me-Phe(4),Gly(5)-ol]-enkephalin (DAMGO; 1 and 2 μg), but not the δ 1-opioid receptor agonist [D-Pen(2,5)]-enkephalin (4 μg) or the δ2-opioid receptor agonist [D-Ala(2),Glu(4)]-deltorphin (2 μg), into the ventral tegmental area (VTA) produced contraversive circling in a dose-dependent manner. The effect of DAMGO was μ-opioid receptor-specific, because the μ-opioid receptor antagonist D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Phe-Thr-NH2 (0.1 and 1 μg), which alone did not elicit any turning behavior, dose-dependently inhibited the effect of DAMGO. Injection of the dopamine D1/D2 receptor antagonist cis-(Z)-flupentixol (1 and 10 μg) into the Nacc shell ipsilaterally to the VTA significantly inhibited DAMGO (2 μg)-induced circling. Similar injections of cis-(Z)-flupentixol into the Nacc core inhibited DAMGO-induced circling, but, in addition, replaced circling by pivoting, namely turning behavior during which the rat rotates around its disfunctioning hindlimb. The present findings show that unilateral stimulation of μ-, but not δ-, opioid receptors in the VTA elicits contraversive circling that requires a relatively hyperdopaminergic activity in both the shell and the core of the Nacc at the opioid-stimulated side of the brain. The Nacc core plays an essential role in the transmission of information directing the display of pivoting that is elicited by an increased dopaminergic activity in the Nacc shell. It is concluded that the Nacc core is an essential link in the feed-forward spiraling striato-nigro-striatal circuitry that transmits information from the Nacc shell toward the DS in freely moving rats.

Topics: Animals; Behavior, Animal; Dopamine; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Male; Motor Activity; Neural Pathways; Nucleus Accumbens; Oligopeptides; Rats; Rats, Wistar; Receptors, Dopamine D1; Receptors, Dopamine D2; Receptors, Opioid, mu

The effect exerted by two gamma-endorphin derivatives (DTgammaE and DEgammaE) was investigated on morphine-induced inhibition on the electrically induced contractions of guinea pig ileum in vitro. Morphine (1x10(-8)-5x10 (-8)-1x10 (-7) M) dose dependently and significantly reduced the E.C. of guinea pig ileum, IC50=6.5x10(-8) M (Confidence limits: 3.7x10 (-8)-9.1x10 (-8)). DTgammaE and DEgammaE per se (1x10 (-6)-5x10 (-6)-1x10 (-5) M) did not modify significantly the E.C. of guinea pig ileum. Furthermore, DTgammaE or DEgammaE injection 10-30-60 min before morphine, did not affect the inhibitory effect of morphine on the E.C. of guinea pig ileum. By contrast, ilea from guinea-pigs treated for 4 days with DTgammaE or DEgammaE (1 mg/Kg/i.p.) were less sensitive to the inhibitory effect of morphine, IC50=8.3x10 (-7) M (Confidence limits: 1.4x10(-6)-3.5x10(-7)) for DTgammaE and IC50=7.7x10(-7) M (Confidence limits: 2.7x10(-6)-8.7x10(-7 )) for DEgammaE . The effect exerted by two beta-endorphin fragments (DTgammaE and DEgammaE) was investigated on the acute opiate withdrawal induced by micro, kappa and delta receptor agonists in vitro. After a exposure in vitro for 4 min to morphine (less selective micro agonist), DAGO (highly selective micro agonist), U50-488H (highly selective kappa agonist) and beta-endorphin (selective micro- delta agonist), a strong contracture of isolated guinea pig ileum was observed after the addition of naloxone. This effect was also observed when isolated rabbit jejunum was pretreated with deltorphin (highly selective delta agonist). DTgammaE or DEgammaE injection before or after treatment with morphine, DAGO, U50-488H, beta-endorphin or deltorphin was able of both preventing and reversing the naloxone-induced contracture after exposure to the opioid agonists in a concentration-dependent fashion. Our results indicate that both DTgammaE or DEgammaE are able to reduce significantly opiate withdrawal in vitro, suggesting an important functional interaction beween beta-endorphin fragments and opioid withdrawal at both micro, kappa and delta receptor level. Our results indicate that chronic treatment of guinea pigs with DTgammaE or DEgammaE induces a significant reduction of the inhibitory effect of morphine on the E.C. of guinea-pig ileum thus confirming an important functional interaction between gamma-endorphin derivatives and opioid system.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Acetylcholine; Animals; beta-Endorphin; Electric Stimulation; Endorphins; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Guinea Pigs; Ileum; In Vitro Techniques; Jejunum; Male; Morphine; Muscle Contraction; Muscle, Smooth; Naloxone; Oligopeptides; Peptide Fragments; Rabbits; Substance Withdrawal Syndrome

To investigate the value of the 2',6'-dimethylphenylalanine (Dmp) residue as an aromatic amino acid substitution, we prepared analogues of the mu opioid receptor-selective dermorphin tetrapeptide Tyr-D-Arg-Phe-betaAla-NH(2) (YRFB) in which Dmp or its D-isomer replaced Tyr(1) or Phe(3). Replacing Phe(3) with Dmp essentially tripled mu receptor affinity and the receptor's in vitro biological activities as determined with the guinea pig ileum (GPI) assay but did not change delta receptor affinity. Despite an inversion of the D configuration at this position, mu receptor affinity and selectivity remained comparable with those of the L-isomer. Replacing the N-terminal Tyr residue with Dmp produced a slightly improved mu receptor affinity and a potent GPI activity, even though the substituted compound lacks the side chain phenolic hydroxyl group at the N-terminal residue. Dual substitution of Dmp for Tyr(1) and Phe(3) produced significantly improved mu receptor affinity and selectivity compared with the singly substituted analogues. Subcutaneous injection of the two analogues, [Dmp(3)]YRFB and [Dmp(1)]YRFB, in mice produced potent analgesic activities that were greater than morphine in the formalin test. These lines of evidence suggest that the Dmp residue would be an effective aromatic amino acid surrogate for both Tyr and Phe in the design and development of novel opioid mimetics.

Topics: Amino Acids, Aromatic; Analgesics, Opioid; Animals; Dose-Response Relationship, Drug; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Guinea Pigs; Ileum; In Vitro Techniques; Indicators and Reagents; Kinetics; Male; Mice; Muscle Contraction; Muscle, Smooth; Oligopeptides; Opioid Peptides; Pain Measurement; Phenylalanine; Receptors, Opioid, mu; Vas Deferens

1 Patch clamp recordings were made from periaqueductal grey (PAG) neurons in vitro to investigate the cellular actions of opioids in wild-type C57B16/J mice and mutant mice lacking the first exon of the micro -opioid (MOP) receptor. 2 In wild-type mice, the kappa-(KOP) agonist U-69593 (300 nM) and the mixed micro /delta-opioid agonist met-enkephalin (10 micro M), but not the delta-(DOP) agonist deltorphin (300 nM), reduced the amplitude of evoked GABA(A)-mediated inhibitory postsynaptic currents (IPSCs). Met-enkephalin and U-69593 also reduced the rate of spontaneous miniature IPSCs, but had no effect on their amplitude and kinetics. In micro -receptor-deleted mice, only U-69593 (300 nM) reduced the amplitude of evoked IPSCs. 3 In wild-type mice, the MOP agonist DAMGO (3 micro M) produced an outward current in 76% of the neurons. Deltorphin and U-69593 produced outward currents in 24 and 32% of the neurons, respectively. In micro -receptor-deleted mice, deltorphin and U-69593 produced similar outward currents in 32 and 27% of the neurons, respectively, while DAMGO was without effect. All neurons in both the wild-type and micro -receptor-deleted mice responded with similar outward currents to either the GABA(B) receptor agonist baclofen (10 micro M), or the opioid-like receptor ORL1 (NOP) agonist nociceptin (300 nM). 4 The DAMGO-, deltorphin-, U-69593-, baclofen- and nociceptin-induced currents displayed inward rectification and reversed polarity at -109 to -116 mV. 5 These findings indicate that micro -, delta- and kappa-opioid receptor activation has complex pre- and postsynaptic actions within the mouse PAG. This differs to the rat PAG where only micro -opioid receptor actions have been observed.

Topics: Analgesics, Opioid; Animals; Benzeneacetamides; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, Methionine; Female; GABA-A Receptor Antagonists; In Vitro Techniques; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Neurons; Oligopeptides; Patch-Clamp Techniques; Periaqueductal Gray; Potassium Channels, Inwardly Rectifying; Pyrrolidines; Receptors, GABA-A; Receptors, Opioid, delta; Receptors, Opioid, kappa; Receptors, Opioid, mu; Synaptic Transmission

The co-abuse of cocaine and ethanol is common among human addicts and has been reported to produce a stronger increase of euphoria as compared to either drug given alone. Both cocaine and ethanol increase the extracellular dopamine concentration in the nucleus accumbens, a terminal region in the mesolimbic dopamine pathway. In addition, both cocaine and ethanol affect the endogenous opioid system, which in turn alters the activity of the mesolimbic dopamine pathway. We have carried out quantitative autoradiography mapping of the opioid receptors as well as the opioid receptor-like 1 receptor in the brains of rats treated with both single and dual cocaine and ethanol. Rats received acute cocaine, ethanol or both drugs in combination. Ethanol alone or in combination with cocaine modulated the receptor densities in rat central nervous system. The kappa receptor densities were generally decreased, while both the mu and the opioid receptor-like 1 receptors were up-regulated. The mu opioid receptor levels were mainly increased in non-cortical regions, whereas the opioid receptor-like 1 receptors were increased in cortical structures. No changes in delta opioid receptors were observed. Cocaine alone did not influence the receptor levels in any of the treatment groups.

Topics: Anesthetics, Local; Animals; Autoradiography; Binding Sites; Brain Mapping; Central Nervous System; Central Nervous System Depressants; Cocaine; Dihydropyridines; Drug Combinations; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Ethanol; Male; Nociceptin Receptor; Oligopeptides; Opioid Peptides; Oximes; Rats; Rats, Sprague-Dawley; Receptors, Opioid; Tritium

The peptide nociceptin/orphanin FQ (N/OFQ) and its receptor ORL-1, also designated opioid receptor 4 (OP(4)) are involved in the modulation of nociception. Using OP(4)-knockout mice, we have studied their response following opioid receptor stimulation and under neuropathic conditions.In vas deferens from wild-type and OP(4)-knockout mice, DAMGO (mu/OP(3) agonist), deltorphine II (delta/OP(1) agonist) and (-)-U-50488 (kappa/OP(2) agonist) induced similar concentration-dependent inhibition of electrically-evoked contractions. Naloxone and naltrindole (delta/OP(1) antagonists) shifted the curves of DAMGO (pA(2)=8.6) and deltorphine II (pA(2)=10.2) to the right, in each group. In the hot-plate assay, N/OFQ (10 nmol per mouse, i.t.) increased baseline latencies two-fold in wild-type mice while morphine (10mg/kg, s.c.), deltorphine II (10 nmol per mouse, i.c.v.) and dynorphin A (20 nmol per mouse, i.c.v.) increased hot-plate latencies by about four- to five-fold with no difference observed between wild-type and knockout mice. Furthermore, no change was evident in the development of the neuropathic condition due to chronic constriction injury (CCI) of the sciatic nerve, after both thermal and mechanical stimulation. Altogether these results suggest that the presence of OP(4) receptor is not crucial for (1) the development of either acute or neuropathic nociceptive responses, and for (2) the regulation of full receptor-mediated responses to opioid agonists, even though compensatory mechanisms could not be excluded.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Analgesics, Non-Narcotic; Analgesics, Opioid; Animals; Dose-Response Relationship, Drug; Dynorphins; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Female; Male; Mice; Mice, Knockout; Morphine; Naloxone; Naltrexone; Narcotic Antagonists; Nociceptin; Nociceptin Receptor; Oligopeptides; Opioid Peptides; Pain; Receptors, Opioid; Time Factors; Vas Deferens

Although transcutaneous electrical nerve stimulation (TENS) is used extensively in inflammatory joint conditions such as arthritis, the underlying mechanisms are unclear. This study aims to demonstrate an opiate-mediated activation of descending inhibitory pathways from the rostral ventral medulla (RVM) in the antihyperalgesia produced by low- (4 Hz) or high-frequency (100 Hz) TENS. Paw withdrawal latency to radiant heat, as an index of secondary hyperalgesia, was recorded before and after knee joint inflammation (induced by intra-articular injection of 3% kaolin and carrageenan) and after TENS/no TENS coadministered with naloxone (20 microg/1 microl), naltrindole (5 microg/1 microl), or vehicle (1 microl) microinjected into the RVM. The selectivity of naloxone and naltrindole doses was tested against the mu-opioid receptor agonist [D-Ala2,N-Me-Phe4,Gly-ol5]-enkephalin (DAMGO) (20 ng, 1 microl) and the delta2-opioid receptor agonist deltorphin (5 microg, 1 microl) in the RVM. Naloxone microinjection into the RVM blocks the antihyperalgesia produced by low frequency (p < 0.001), but not that produced by high-frequency TENS (p > 0.05). In contrast, naltrindole injection into the RVM blocks the antihyperalgesia produced by high-frequency (p < 0.05), but not low-frequency (p > 0.05) TENS. The analgesia produced by DAMGO and deltorphin is selectively blocked by naloxone (p < 0.05) and naltrindole (p < 0.05), respectively. Thus, the dose of naloxone and naltrindole used in the current study blocks mu- and delta-opioid receptors, respectively. Hence, low-frequency and high-frequency TENS produces antihyperalgesia by activation of mu- and delta-opioid receptors, respectively, in the RVM.

Topics: Analgesics, Opioid; Animals; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Hot Temperature; Hyperalgesia; Male; Medulla Oblongata; Naloxone; Naltrexone; Narcotic Antagonists; Oligopeptides; Pain Measurement; Rats; Rats, Sprague-Dawley; Receptors, Opioid; Receptors, Opioid, delta; Receptors, Opioid, mu; Transcutaneous Electric Nerve Stimulation

kappa-opioid receptor agonists (kappa-ORAs) have been shown to modulate visceral nociception through an interaction with a peripheral, possibly novel, kappa-opioid-like receptor. We used in the present experiments an antisense strategy to further explore the hypothesis that kappa-ORA effects in the colon are produced at a site different from the cloned kappa-opioid receptor (KOR). An antisense oligodeoxynucleotide (ODN) to the cloned rat KOR was administered intrathecally (12.5 microg, twice daily for 4 d) to specifically knock-down the cloned KOR. Efficacy of the KOR antisense ODN treatment was behaviorally evaluated by assessing the antinociceptive effects of peripherally administered kappa- (EMD 61, 753 and U 69,593), mu- (DAMGO) and delta- (deltorphin) ORAs in the formalin test. Intrathecal antisense, but not mismatch ODN blocked the actions of EMD 61,753 and U 69,593 without affecting the actions of DAMGO or deltorphin; a complete recovery of antinociceptive actions of the kappa-ORA EMD 61,753 was observed 10 d after the termination of antisense ODN treatment. In contrast, the ability of EMD 61,753 to dose-dependently attenuate responses of pelvic nerve afferent fibers to noxious colonic distension was unaffected in the same rats in which the antisense ODN effectively knocked-down the KOR as assessed in the formalin test. Additionally, Western blot analysis demonstrated a significant downregulation of KOR protein in the L4-S1 dorsal root ganglia of antisense, but not mismatch ODN-treated rats. The present results support the existence of a non-kappa-opioid receptor site of action localized in the colon.

Topics: Acetamides; Analgesics, Opioid; Animals; Colon; Disinfectants; Electrophysiology; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Formaldehyde; Ganglia, Spinal; Male; Nociceptors; Oligonucleotides, Antisense; Oligopeptides; Pain; Physical Stimulation; Pyrrolidines; Rats; Rats, Sprague-Dawley; Receptors, Opioid, kappa; Rectum; Visceral Afferents

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

delta-Opioid receptors have been implicated in reinforcement processes and antagonists are available that produce long-lasting and selective antagonism of delta-opioid receptors in vivo. This experiment assessed the contribution of delta-opioid receptors to the antinociceptive and reinforcing properties of heroin. The effects of the irreversible delta-antagonist naltrindole-5'-isothiocyanate (5'-NTII) were evaluated on heroin self-administration and hot-plate antinociception in rats. 5'-NTII (10 nmol i.c.v.) shifted the dose-response curve for heroin self-administration downward, increasing the A(50) values on the ascending and descending limbs by approximately 0.5 log units and decreasing the maximum by 33%. 5'-NTII (40 nmol i.c.v.) shifted both limbs of the heroin self-administration dose-effect curve 1.2 log units to the right and decreased the maximum by 90%. Heroin self-administration gradually returned to baseline levels over 7 or 17 days after administration of 10 or 40 nmol 5'-NTII, respectively. 5'-NTII (40 nmol i.c.v.) decreased the self-administration of 0.17 mg/infusion cocaine by 40% while having no effect on responding maintained by 0.33 or 0.67 mg/infusion. 5'-NTII attenuated the antinociceptive effects of deltorphin (delta(2)) in a dose-dependent manner while having no effect on antinociception elicited after i.c. v. administration of [D-Pen(2),D-Pen(5)]-enkephalin (delta(1)) or [D-Ala(2),N-Me-Phe(4),Gly(5)-ol]-enkephalin (mu). In addition, the antinociceptive effects of heroin were not significantly affected by 5'-NTII (40 nmol i.c.v.). Therefore, 5'-NTII can attenuate the reinforcing effects of heroin at doses that do not affect its antinociceptive effects. Long-acting delta(2)-opioid antagonists may be beneficial in the treatment of heroin dependence or as adjuncts to reduce the abuse liability of opioid analgesics.

Topics: Analgesics, Opioid; Animals; Cocaine; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Heroin; Injections, Intraventricular; Isothiocyanates; Male; Naltrexone; Narcotic Antagonists; Oligopeptides; Pain Measurement; Rats; Rats, Inbred F344; Receptors, Opioid, delta; Reinforcement, Psychology; Self Administration

Opioid receptor subtypes, delta and kappa, are found in cardiac tissue and may play a role in cardiac function. We explored if the synthetic opioid delta(2)[D-Ala(2)]-deltorphin (DTP) and mu peptide agonist [D-Ala(2)]-enkephalin (DAMGO) alter the left ventricular pressure (LVP) [Ca(2+)](i) relationship in isolated guinea pig hearts. LV phasic [Ca(2+)](i) was measured from dual fluorescence signals using indo 1. Ca(2+) transients were corrected and calibrated to nM [Ca(2+)](i). Diastolic (d), systolic (s) [Ca(2+)](i), and s-d[Ca(2+)](i) were plotted v LVP at 0.3 to 6.8 mM [CaCl(2)](e)to assess the association of contractility to Ca(2+). Also given were naltriben (NTB) and CTOP, delta(2) and mu antagonists, and nifedipine (NIF) and thapsigargin (THAP). From a control of 880+/-95 nM (SEM), DTP decreased s-d[Ca(2+)](max) to 525+/-82 nM after DTP and to 405+/-84 nM after NIF, whereas THAP increased s-d[Ca(2+)](max)to 1605+/-275 nM. NTB, 795+/-33 nM, NTB+DTP, 820+/-98 nM, DAMGO, 970+/-82 nM, and DAMGO+CTOP, 830+/-93 nM, gave values similar to controls. From a control value of 61+/-4 mm Hg, LVP(max)was increased by DTP to 73+/-3 mmHg and by THAP to 77+/-2 mmHg, was unchanged by DAMGO at 48+/-6 mmHg, and was decreased by NIF to 24+/-2 mmHg. Compared to the control value of 594+/-18 nM, less s-d[Ca(2+)](i) was required to attain 50% s-dLVP(max)(curve left shift) with increasing [CaCl(2)](e) for DTP, 407+/-17 nM, and more was required for THAP, 737+/-35 nM. DTP raised the slope max of s-dLVP(max)(100%) v. s-d[Ca(2+)](i)by 2.7-fold. This indicates DTP enhances cardiac performance by enhancing responsiveness to cytosolic Ca(2+)rather than by raising diastolic Ca(2+) and subsequently released Ca(2+), as does THAP.

Topics: Animals; Calcium; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Guinea Pigs; Heart; Myocardial Contraction; Oligopeptides; Receptors, Opioid, delta; Signal Transduction

The nucleus accumbens, and particularly its shell region, is a critical site at which feeding responses can be elicited following direct administration of opiate drugs as well as micro-selective and delta-selective, but not kappa-selective opioid receptor subtype agonists. In contrast to observations of selective and receptor-specific opioid antagonist effects upon corresponding agonist-induced actions in analgesic studies, ventricular administration of opioid receptor subtype antagonists blocks feeding induced by multiple opioid receptor subtype agonists. The present study examined whether feeding responses elicited by either putative mu ([D-Ala(2), NMe-Phe(4), Gly-ol(5)]-enkephalin (DAMGO)), delta(1) ([D-Pen(2), D-Pen(5)]-enkephalin (DPDPE)) or delta(2) ([D-Ala(2), Glu(4)]-deltorphin (Deltorphin)) opioid receptor subtype agonists administered into the nucleus accumbens shell were altered by accumbens pretreatment with either selective mu (beta-funaltrexamine), mu(1) (naloxonazine), delta(1) ([D-Ala(2), Leu(5), Cys(6)]-enkephalin (DALCE)), delta(2) (naltrindole isothiocyanate) or kappa(1) (nor-binaltorphamine) opioid receptor subtype antagonists. Similar magnitudes and durations of feeding responses were elicited by bilateral accumbens administration of either DAMGO (2.5 microg), DPDPE (5 microg) or Deltorphin (5 microg). DAMGO-induced feeding in the nucleus accumbens shell was significantly reduced by accumbens pretreatment of mu, delta(1), delta(2) and kappa(1), but not mu(1) opioid receptor subtype antagonists. DPDPE-induced feeding in the accumbens was significantly reduced by accumbens pretreatment of mu, delta(1), delta(2) and kappa(1), but not mu(1) opioid receptor subtype antagonists. Deltorphin-induced feeding in the accumbens was largely unaffected by accumbens delta(2) antagonist pretreatment, and was significantly enhanced by accumbens mu or kappa(1) antagonist pretreatment. These data indicate different opioid pharmacological profiles for feeding induced by putative mu, delta(1) and delta(2) opioid agonists in the nucleus accumbens shell, as well as the participation of multiple opioid receptor subtypes in the elicitation and maintenance of feeding by these agonists in the nucleus accumbens shell.

Topics: Animals; Eating; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Male; Nucleus Accumbens; Oligopeptides; Rats; Rats, Sprague-Dawley; Receptors, Opioid, delta; Receptors, Opioid, mu

The nucleus accumbens (NAcc) has been implicated as an important reward site for the mediation of unconditioned reinforcers such as food. Although both mu-selective and delta-selective opioid agonists in the NAcc induce spontaneous and palatable feeding, these effects are mediated by multiple opioid receptor subtypes within the nucleus. A role for dopaminergic mediation of feeding in the NAcc is based upon selective antagonist-induced suppression of feeding induced by systemic amphetamine. The present study investigated whether feeding elicited by infusion of either mu ([D-Ala(2), NMe-Phe(4), Gly-ol(5)]-enkephalin) or delta(2) ([D-Ala(2), Glu(4)]-deltorphin) opioid receptor subtype agonists in the shell region of the NAcc would be modified by intra-accumbens pretreatment with equimolar (12-100 nmol) doses of either D(1)-selective (SCH23390) or D(2)-selective (raclopride) antagonists. Both opioid agonists displayed comparable magnitudes and durations of feeding responses in the NAcc. SCH23390 significantly and dose-dependently reduced mu agonist-induced feeding in the NAcc with significant reductions noted following the two higher, but not two lower doses. In contrast, raclopride pretreatment produced inconsistent effects upon mu agonist-induced feeding with limited actions across doses and test times. Further, neither SCH23390 nor raclopride pretreatment in the NAcc affected feeding elicited by the delta(2) opioid agonist. These data indicate that the role of dopamine receptors in mediating opioid-induced feeding within the shell region of the NAcc is both dependent upon the dopamine receptor subtype that was blocked (D(1) vs. D(2)) as well as the opioid receptor subtype which was being stimulated mu vs. delta(2)).

Topics: Analgesics, Opioid; Animals; Dopamine Antagonists; Eating; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Male; Nucleus Accumbens; Oligopeptides; Rats; Rats, Sprague-Dawley; Receptors, Dopamine D1; Receptors, Dopamine D2; Receptors, Opioid, delta; Receptors, Opioid, mu

Projection neurons of the striatum release opioid peptides in addition to GABA. Our previous studies showed that the opioid peptide dynorphin regulates that subtype of projection neurons which sends axons to the substantia nigra/entopeduncular nucleus, as indicated by an inhibitory action of dynorphin/agonists on D1 dopamine receptor-mediated immediate-early gene induction in these neurons. The other subtype of striatal projection neurons projects to the globus pallidus and contains the opioid peptide enkephalin. Here, we investigated whether enkephalin regulates the function of striatopallidal neurons, by analysing opioid effects on immediate-early gene induction by D2 dopamine receptor blockade that occurs in these neurons. Thus, the effects of systemic and intrastriatal administration of various opioid receptor agonists and antagonists on immediate-early gene expression (c-fos, zif 268) induced by the D2 receptor antagonist eticlopride were examined with in situ hybridization histochemistry. Intrastriatal infusion of enkephalin (delta and mu), but not dynorphin (kappa), receptor agonists suppressed immediate-early gene induction by eticlopride in a dose-dependent manner. This suppression was blocked by the opioid receptor antagonist naloxone, confirming the involvement of opioid receptors. Repeated treatment with D2 receptor antagonists produces increased enkephalin expression and diminished immediate-early gene inducibility in striatopallidal neurons, as well as behavioral effects that are attenuated compared to those of acute treatment (e.g., reduced akinesia). Naloxone reversed such behavioral recovery (i.e. reinstated akinesia), but did not significantly affect suppressed immediate-early gene induction. Our results indicate that enkephalin acts, via mu and delta receptors in the striatum, to inhibit acute effects of D2 receptor blockade in striatopallidal neurons. Moreover, the present findings suggest that increased enkephalin expression after repeated D2 receptor antagonist treatment is an adaptive response that counteracts functional consequences of D2 receptor blockade, but is not involved in suppressed immediate-early gene induction. Together with our earlier findings of the role of dynorphin, these results indicate that opioid peptides in the striatum serve as negative feedback systems to regulate the striatal output pathways in which they are expressed.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Analgesics; Animals; Base Sequence; Corpus Striatum; DNA Primers; DNA-Binding Proteins; Dopamine Antagonists; Dopamine D2 Receptor Antagonists; Early Growth Response Protein 1; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, Leucine-2-Alanine; Enkephalins; Gene Expression Regulation; Genes, fos; Genes, Immediate-Early; Immediate-Early Proteins; In Situ Hybridization; Male; Molecular Sequence Data; Naloxone; Neurons; Oligopeptides; Proto-Oncogene Proteins c-fos; Rats; Rats, Sprague-Dawley; Salicylamides; Transcription Factors; Transcriptional Activation

The effects of phospholipase A2, cyclooxygenase-1, cyclooxygenase-2, and 5-lipoxygenase inhibitors on acute opiate withdrawal induced by selective mu, kappa and delta receptor agonists was investigated in vitro. After a 4 min in vitro exposure to D-Ala2-N-methyl-Phe-Gly5-ol)enkephalin (DAMGO; a highly selective mu agonist) and trans(+/-)-3,4-dichloro-N-methyl-N-(2(1pyrrolidynyl)-cyclohexyl)-+ ++benzeneacetamid (U50-488H; a highly selective K agonist) a strong contraction of the guinea pig isolated ileum was observed after the addition of naloxone. This effect was also observed when rabbit isolated jejunum was pretreated with deltorphin (a highly selective delta agonist). Mepacrine (a phospholipase A2 inhibitor), tolmetin (a selective cyclooxygenase-1 inhibitor) and meloxicam (a selective cyclooxygenase-2 inhibitor) treatment before or after DAMGO or U50-488H were able to both prevent and reverse the naloxone-induced contraction after exposure to the opioid agonists, in a concentration-dependent fashion. In addition, nordihydroguaiaretic acid (a 5-lipooxygenase inhibitor) was able to block the naloxone-induced contraction following exposure to DAMGO or U50-488H if injected either before or after the opioid agonist. In contrast, mepacrine, tolmetin, meloxicam and nordihydroguaiaretic acid did not affect the naloxone-induced contraction after exposure to deltorphin. The results of the present study confirm and extend a previous study performed with morphine indicating that arachidonic acid and its metabolites (prostaglandins and leukotrienes) are involved in the development of opioid withdrawal induced by selective mu and kappa opioid agonists whereas no effects were observed on withdrawal induced by the selective delta opioid agonist deltorphin.

Topics: Animals; Arachidonate 5-Lipoxygenase; Arachidonic Acid; Benzeneacetamides; Cyclooxygenase 1; Cyclooxygenase 2; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Enzyme Inhibitors; Guinea Pigs; Ileum; In Vitro Techniques; Isoenzymes; Jejunum; Male; Oligopeptides; Opioid-Related Disorders; Phospholipases A; Phospholipases A2; Prostaglandin-Endoperoxide Synthases; Pyrroles; Receptors, Opioid, delta; Receptors, Opioid, kappa; Receptors, Opioid, mu

Most electrophysiological studies of opioids on hippocampal principal neurons have found indirect actions, usually through interneurons. However, our laboratory recently found reciprocal alteration of the voltage-dependent K(+) current, known as the M-current (I(M)), by kappa and delta opioid agonists in CA3 pyramidal neurons. Recent ultrastructural studies have revealed postsynaptic delta opiate receptors on dendrites and cell bodies of CA1 and CA3 hippocampal pyramidal neurons (HPNs). Reasoning that previous electrophysiological studies may have overlooked voltage-dependent postsynaptic effects of the opioids in CA1, we reevaluated their role in CA1 HPNs using the rat hippocampal slice preparation for intracellular current- and voltage-clamp recording. None of the delta and mu; receptor-selective opioids tested, including [D-Pen(2,5)]-enkephalin (DPDPE), [D-Ala(2)]-deltorphin II (deltorphin), [D-Ala(2), NMe-Phe(4), Gly-ol]-enkephalin (DAMGO), and [D-Ala(2), D-Leu(5)] enkephalin (DADLE), altered membrane properties such as I(M) or Ca(2+)-dependent spikes in CA1 HPNs. The nonopioid, Des-Tyr-dynorphin (D-T-dyn), also had no effect. By contrast, dynorphin A (1-17) markedly increased I(M) at low concentrations and caused an outward current at depolarized membrane potentials. The opioid antagonist naloxone and the kappa receptor antagonist nor-binaltorphimine (nBNI) blocked the I(M) effect. However, the kappa-selective agonists U69,593 and U50,488h did not significantly alter I(M) amplitudes when averaged over all cells tested, although occasional cells showed an I(M) increase with U50,488h. Our results suggest that dynorphin A postsynaptically modulates the excitability of CA1 HPNs through opiate receptors linked to voltage-dependent K(+) channels. These findings also provide pharmacological evidence for a functional kappa opiate receptor subtype in rat CA1 HPNs but leave unanswered questions on the role of delta receptors in CA1 HPNs.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Animals; Benzeneacetamides; Dynorphins; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalin, Leucine-2-Alanine; Hippocampus; In Vitro Techniques; Oligopeptides; Peptide Fragments; Potassium Channels; Pyramidal Cells; Pyrrolidines; Rats; Receptors, Opioid; Receptors, Opioid, delta; Receptors, Opioid, kappa

Topics: Analgesics; Animals; Depression, Chemical; Disulfides; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Enzyme Inhibitors; Male; Motor Activity; Neprilysin; Oligopeptides; Pain Measurement; Peptides; Phenylalanine; Rats; Rats, Inbred F344; Receptors, Opioid; Receptors, Opioid, delta; Receptors, Opioid, kappa; Receptors, Opioid, mu

High affinity low K(m) GTPase activity was measured in membrane preparations of adult mouse mesencephalic periaqueductal grey matter (PAG). Opioids displaying selectivity towards mu- or delta-opioid receptors (OR) activated the enzyme in a concentration-dependent manner. Antibodies to mu-OR greatly impaired the potential of mu-agonists, [D-Ala2,N-MePhe4,Gly-ol5]-enkephalin (DAMGO) and morphine, to increase hydrolysis of GTP. The same antibodies had little effect on [D-Pen2,5]enkephalin (DPDPE) and [D-Ala2]deltorphin II, both agonists at delta-OR. Stimulation of GTPase by DPDPE and [D-Ala2]deltorphin II - but not by morphine or DAMGO - was diminished by antibodies to delta-OR. The blockade of G(i2)alpha subunits by specific antibodies impaired the activation of G alpha-related GTPase by all opioids. Antibodies in vitro, and oligodeoxynucleotides in vivo, prepared against Gx/z alpha subunits, reduced the release of Pi promoted by DAMGO and morphine. The impairment of Gx/z proteins also slightly reduced the effect of the delta2 agonist [D-Ala2]deltorphin II. At delta1 receptors, DPDPE fully expressed its activation of GTPase. These results indicate that in the PAG, mu-OR and delta-OR couple with Gi2 transducer proteins. Notably, mu-OR also regulates the pertussis toxin-insensitive G-protein Gx/z, an effect poorly exhibited by delta-OR in this tissue.

Topics: Animals; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalins; GTP Phosphohydrolases; GTP-Binding Proteins; Immunochemistry; Injections, Intraventricular; Kinetics; Male; Mice; Oligonucleotides; Oligopeptides; Periaqueductal Gray; Receptors, Opioid, delta; Receptors, Opioid, mu; Stimulation, Chemical

The solid phase procedure, based on the Fmoc chemistry, was used to prepare some opioid deltorphin (H-Tyr-D-Ala-Phe-Asp-Val-Val-Gly-NH2, DEL C) and dermorphin (H-Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2, DER) analogues in which a D-glucopyranosyl moiety is beta-O-glycosidically linked to a Thr4 or Thr7 side chain. Their activities were determined in binding studies based on displacement of mu- and delta-receptor selective radiolabels from rat brain membrane synaptosomes, in guinea pig ileum and rabbit jejenum bioassays, and, in vivo, by a mouse tail-flick test after intracerebroventricular (icv) and subcutaneous (sc) administrations. The glyco analogues modified at position 4 displayed low opioid properties, while Thr7-glycosylated peptides retained high delta- or mu-selectivity and remarkable activity in vivo. In particular, as systemic antinociceptive agents, the latter glucoside-bearing compounds were more potent than the parent unglycosylated peptide counterparts, showing a high blood to brain rate of influx which may be due to the glucose transporter GLUT-1.

Topics: Amino Acid Sequence; Analgesics, Opioid; Animals; Binding, Competitive; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Glycopeptides; Guinea Pigs; Ileum; In Vitro Techniques; Indicators and Reagents; Jejunum; Kinetics; Male; Mice; Muscle Contraction; Muscle, Smooth; Oligopeptides; Opioid Peptides; Pain; Rabbits; Rats; Rats, Sprague-Dawley; Receptors, Opioid; Structure-Activity Relationship; Synaptosomes

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

Microinjection of [D-Ala2,MePhe4,Gly(ol)5]enkephalin (DAMGO) into either the periaqueductal gray (PAG) or the rostral ventral medulla (RVM) elicits analgesia in the tailflick assay in the rat. Co-administration of DAMGO into both regions together results in a profound synergistic interaction similar to that we previously reported with morphine. U50,488H and DPDPE are inactive when given into either region. [D-Ala2,Glu4]Deltorphin (deltorphin), on the other hand, elicits an analgesic response, although the maximal response is less than than mu agonists. Co-administration of DAMGO into one region with deltorphin in the other also results in a significant synergy. However, co-administration of DAMGO and deltorphin together in the same region gives only additive effects. These results confirm the existence of mu/mu synergy between the PAG and RVM. kappa 1 and delta 1 agents are inactive, but the delta 2 agonist deltorphin is active in both regions. Our results indicate the presence of mu/delta 2 synergy between the PAG and RVM which appears to involve interactions of pathways rather than receptor interactions at the cellular level.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Amino Acid Sequence; Analgesics; Animals; Drug Synergism; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalins; Male; Medulla Oblongata; Molecular Sequence Data; Oligopeptides; Pain; Periaqueductal Gray; Pyrrolidines; Rats; Rats, Sprague-Dawley; Receptors, Opioid, delta; Receptors, Opioid, mu

In this work, we have characterized the opioid receptor expressed by the human neuroblastoma cell line SK-N-BE and compared its hydrodynamic behaviour with those of well known opioid receptors: mu-opioid receptor of rabbit cerebellum and delta-opioid receptor of the hybrid cell line NG 108-15. Human neuroblastoma cell line SK-N-BE expresses a substantial amount of opioid receptors (200-300 fmoles/mg of protein). Pharmacological characterization suggests an heterogenous population of receptors and the presence of two delta subtypes which are, at least partially, negatively coupled with adenylate cyclase via a Gi protein. These receptors exist under two different molecular forms and, in this respect, strikingly contrast with the archetypic delta receptors of NG 108-15 hybrid cell line which show only a high molecular weight form and appear more tightly coupled with the G protein. Hydrodynamic behaviour of SK-N-BE opioid receptors is reminiscent of the profile observed with the rabbit cerebellum mu-opioid receptor. This observation is consistent with the presence of two delta-opioid receptors subtypes, one of which exhibiting properties close to those of mu opioid receptors. Taken overall, our results suggest that different types and subtypes of opioid receptors, even if they are coupled to the same inhibitory G protein, are more or less tightly coupled with their transduction proteins and that closely related opioid receptors can form allosterically interacting complexes.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Adenylyl Cyclases; Amino Acid Sequence; Animals; Binding, Competitive; Cerebellum; Colforsin; Diprenorphine; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalins; Etorphine; GTP-Binding Proteins; Guinea Pigs; Humans; Hybrid Cells; Molecular Sequence Data; Morphine; Naloxone; Neoplasm Proteins; Nerve Tissue Proteins; Neuroblastoma; Oligopeptides; Pyrrolidines; Rabbits; Receptors, Opioid; Receptors, Opioid, delta; Receptors, Opioid, mu; Tumor Cells, Cultured

This study characterized the antinociception produced by intrathecal (i.t.) administration of the respective delta-2 and delta-1 receptor-selective agonists, [D-Ala2, Glu4]deltorphin (DELT) and DPDPE or the mu receptor selective agonist DAMGO in the rat. It also determined whether the antinociception produced by these opioid agonists was differentially affected by i.t. coadministration of the delta-2 receptor-selective antagonist, naltriben (NTB). In the tail-flick test, the ED50 values of DELT and DPDPE were 2.7 micrograms (3.4 nmol) and 19.0 micrograms (29.4 nmol), respectively. Coadministration of 3 micrograms (6.4 nmol) of NTB increased the ED50 of DELT at least 25-fold, but did not significantly increase the ED50 of DPDPE. These findings suggest that: 1) DELT and DPDPE act at different delta opioid receptor subtypes in the rat spinal cord; 2) 3 micrograms of NTB can distinguish these receptor subtypes and 3) activation of either delta-1 or delta-2 receptors is sufficient to produce antinociception in the tail-flick test. Although NTB did not antagonize the increase in tail-flick latency produced by 0.1 to 0.3 microgram of DAMGO, it did antagonize the increase produced by 0.03 microgram of DAMGO resulting in a steeper dose-response relationship. Unlike DPDPE or DAMGO, DELT did not increase hot-plate latency except at a dose that produced adverse motor effects. Coadministration of 3 micrograms of NTB antagonized the increase in hot-plate latency produced by DPDPE, but not DAMGO, suggesting that this delta-1 receptor-selective agonist may also have efficacy at delta-2 receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Dose-Response Relationship, Drug; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalins; Injections, Spinal; Male; Naltrexone; Narcotic Antagonists; Oligopeptides; Rats; Rats, Sprague-Dawley; Receptors, Opioid, delta; Receptors, Opioid, mu; Reflex; Spinal Cord

Conformationally restricted deltorphin analogues were synthesized either through incorporation of cyclic phenylalanine analogues in position 2 or 3 of the peptide sequence or through various side chain-to-side chain cyclizations. Compounds were tested in mu-, delta-, and kappa-receptor selective binding assays and in the guinea pig ileum (GPI) and mouse vas deferens (MVD) bioassays. Replacement of Phe3 in [D-Ala2]deltorphin I with 2-aminoindan-2-carboxylic acid (Aic) or L- or D-2-aminotetralin-2-carboxylic acid (Atc) resulted in agonist compounds which retained the high delta receptor selectivity of the parent peptide. Substitution of a tetrahydroisoquinoline-3-carboxylic acid (Tic) residue in the 2-position of [D-Ala2]deltorphin I and of [Phe4,Nle6]deltorphin produced a partial delta agonist, H-Tyr-Tic-Phe-Asp-Val-Val-Gly-NH2, and a pure delta antagonist, H-Tyr-Tic-Phe-Phe-Leu-Nle-Asp-NH2, respectively. The latter antagonist displayed high delta selectivity (Ki mu/Ki delta = 502) and was a potent antagonist against selective delta agonists in the MVD assay (Ke congruent to 10 nM). Various [D-Ala2]-deltorphin I analogues cyclized between the side chains of Orn (or Lys) and Asp (or Glu) residues substituted in positions 2 and 4, 4 and 7, and 2 and 7 were essentially nonselective. Comparison with corresponding N-terminal tetrapeptide analogues revealed that the C-terminal tripeptide segment in the deltorphin heptapeptides made a crucial contribution to delta affinity and delta selectivity in the case of the agonist peptides but not in the case of the antagonist.

Topics: Amino Acid Sequence; Animals; Binding Sites; Brain; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, Leucine; Enkephalins; Guinea Pigs; Ileum; In Vitro Techniques; Male; Mice; Molecular Sequence Data; Oligopeptides; Protein Conformation; Receptors, Opioid, delta; Receptors, Opioid, kappa; Receptors, Opioid, mu; Vas Deferens

Deltorphins A, B, and C exhibit high delta-affinities (Ki = 0.12-0.31 nM) and very high delta-receptor binding selectivities (Ki mu/Ki delta = 1800-4100). A study of the delta-receptor binding properties of 15 deltorphin analogues focused primarily on the influence of the anionic group in the C-terminal tetrapeptide. Amidation of the beta-carboxyl groups of [Asp7], [Glu4], or [Asp4] in deltorphins A, B, and C, respectively, yielded peptides with enhanced mu-receptor affinities and minor changes in delta-affinities (Ki delta = 0.20-0.65 nM), but 5-8-fold diminished delta-selectivities. A free C-terminal carboxyl group markedly reduced delta-affinities and decreased delta-selectivities 6-11-fold; thus, the C-terminal amide group critically facilitates delta-affinity. Modifications in the anionic charged group or hydrophobic residues in the C-terminal tetrapeptide address domain of deltorphin A altered spatial distributions critical for delta-affinity and selectivity; e.g., [Nle6]deltorphin A enhanced mu-affinity and lowered delta-selectivity by two-thirds; the progressive, step-wise repositioning of Asp in deltorphin C (from position 4 to 7) was accompanied by linear decreases in delta-affinities and -selectivities, and increased mu-affinities of these peptides; enhancement of the charge density to -2, in [Asp6, Asp-OH7]deltorphin A, decreased delta-affinity and -selectivity, while [Asp4,5,His7]deltorphin A bound to neither mu- nor delta-sites. These results demonstrate that while an anionic group may occasionally facilitate high delta-receptor affinity, it represents an absolute requirement for the high delta-binding selectivity of these peptides. The locations of the charged groups relative to hydrophobic residues in the address domain of the peptide are also critical determinants for both delta-affinity and -selectivity.

Topics: Animals; Binding, Competitive; Brain; Cell Membrane; Enkephalin, Leucine-2-Alanine; Oligopeptides; Rats; Receptors, Opioid; Receptors, Opioid, kappa; Receptors, Opioid, mu; Structure-Activity Relationship

Deltorphin N-terminal tetrapeptides [DEL A: H-Tyr-D-Met-Phe-His-R, where R = -NH2, -NH-NH2, -OCH3, -OH, -NH-NH-CO-R' (R' = -CH3 or adamantane); DEL C: H-Tyr-D-Ala-Asp-R (R = -OH, -NHCH3)], were used in a receptor binding assay with [3H]DADLE and [3H]DPDPE for delta sites, and [3H]DAGO for mu sites; tetrapeptide Ki delta values were similar with either [3H]-delta ligand. DEL A tetrapeptides C-terminally substituted with -NH2, -NH-NH2, -OCH3, and -OH had 10 to greater than 1,000-fold decreased Ki delta values, while Ki mu increased 5 to 100-fold to yield mu selectivity. C-Terminal substitution with -NH-NH2 and -OCH3 conferred highest mu selectivities; adamantyl and acetyl hydrazide derivatives were non-selective. DEL-(1-4)-OH peptides had decreased delta and mu affinities: DEL A-[Asp4]-(1-4)-OH and DEL C-(1-4)-OH had low affinities (greater than 1 microM), however, the Ki delta of the former was 5-fold greater than the latter, and the Ki mu was less by 15-fold. The data suggest that the "message" domain of DEL exhibits receptor selectivity different from that of the heptapeptide.

Topics: Animals; Brain; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalin, Leucine-2-Alanine; Enkephalins; Kinetics; Oligopeptides; Rats; Receptors, Opioid; Receptors, Opioid, delta; Receptors, Opioid, mu; Structure-Activity Relationship; Synaptosomes

Dermorphin, Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2 is an extraordinarily potent and highly mu-selective opioid heptapeptide isolated from amphibian skin. It is unique among peptides synthesized by animal cells in having an amino acid residue in the D-configuration. At least two different preprodermorphin cDNAs were cloned from skin of Phylomedusa sauvagei; their predicted amino acid sequences contained four to five homologous repeats of 35 amino acids, each repeat including one copy of the dermorphin progenitor sequence. Tyr-Ala-Phe-Gly-Tyr-Pro-Ser-Gly, flanked by Lys-Arg at the amino end and by Glu-Ala-Lys-Lys at the carboxyl end [Science (Wash. D. C.) 238:200-202 (1987)]. The D-Ala in position 2 in dermorphin is encoded by a usual Ala codon in the precursor sequence. Of the two prodermorphin molecules, one has a dermorphin copy replaced with a distinct heptapeptide same processing signals. Assuming the same pathway as for the release of dermorphin, processing of this precursor may yield, beside dermorphin, a copy of a new peptide, Tyr-D-Met-Phe-His-Leu-Met-Asp-NH2. We have synthetized this peptide together with its (L-Met2)-counterpart and evaluated their respective opioid receptor selectivity in the mouse vas deferens and guinea pig ileum assays and in rat brain membrane binding assays. Overall, the data collected demonstrate that the putative prodermorphin product Tyr-D-Met-Phe-His-Leu-Met-Asp-NH2 named dermenkephalin, behaves as a potent delta opioid agonist exhibiting high affinity and high selectivity for the delta opioid receptor. Prodermorphin, thus, offers a surprising example of an opioid biosynthetic precursor that might simultaneously generate highly potent and fully selective agonists for the mu- (morphine) and the delta (enkephalin) opioid receptors, respectively. In addition, because dermenkephalin has no structural features in common with the sequence of all the hitherto known opioid peptides, it should be a useful tool for identifying conformational determinants for high affinity and selective binding of opioids to the delta receptor.

Topics: Amino Acid Sequence; Analgesia; Animals; Binding, Competitive; Brain; Cell Membrane; Cerebral Ventricles; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalins; Injections, Intraventricular; Kinetics; Male; Mice; Oligopeptides; Opioid Peptides; Pain; Rats; Receptors, Opioid; Receptors, Opioid, delta