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

The partial mu-opioid receptor pool inactivation strategy in isolated mouse vas deferens was used to determine partial agonism of endomorphins and their analogs (endomorphin-1-ol, 2',6'-dimethyltyrosine (Dmt)-endomorphin-1, endomorphin-2-ol and (D-Met2)-endomorphin-2) using morphine, normorphine, morphiceptin, (D-Ala2,MePhe4,Gly5-ol)-enkephalin (DAMGO) and its amide (DAMGA) as reference opioid agonists. Agonist affinities (KA) and efficacies were assessed both by the "null" and the "operational" method. The KA values determined by the two methods correlated significantly with each other and also with the displacing potencies against 3H-naloxone in the receptor binding assay in the presence of Na+. DAMGO and DAMGA were full agonist prototypes, morphine, endomorphin-1, endomorphin-1-ol, Dmt-endomorphin-1, endomorphin-2-ol and (D-Met2)-endomorphin-2 were found by both methods to be partial agonists whereas the parameters for normorphine, morphiceptin and endomorphin-2 were intermediate.

Topics: Animals; Brain; Dose-Response Relationship, Drug; Endorphins; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Male; Mice; Morphine Derivatives; Naloxone; Oligopeptides; Rats; Receptors, Opioid, mu; Vas Deferens

The kinetic parameters of antagonism by the delta opioid receptor selective antagonist N-t-Boc-Tyr-Pro-Gly-Phe-Leu-Thr, obtained by using moderately selective or selective agonists, were compared in the mouse vas deferens bioassay. The apparent affinity for the preferred receptor type was 6.8 times higher when selective agonist was used, resulting in a Ke of 81.4 nM (66.3-99.9, n = 6) against [D-Ala2, D-Leu5]-enkephalin, with a 3700-fold delta over mu or kappa selectivity ratio.

Topics: Analgesics; Animals; Anticonvulsants; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, Leucine-2-Alanine; Enkephalin, Methionine; Enkephalins; In Vitro Techniques; Kinetics; Male; Mice; Mice, Inbred Strains; Morphine Derivatives; Naltrexone; Oligopeptides; Pyrroles; Receptors, Opioid, delta; Thiophenes; Vas Deferens

[3H][D-Pen2, D-Pen5]enkephalin binding to intact NG 108-15 cells has been measured under physiological conditions of temperature and medium. The dissociation constant (7.78 nM), receptor density (385 fmol/mg protein), and Hill slope (1.06) values measured under these conditions are consistent with values obtained by others using membranes prepared from these cells. Kinetic analysis of the radioligand binding to these cells show biphasic association and monophasic dissociation processes suggesting the presence of different receptor affinity states for the agonist. The data show that the binding affinity of [3H][D-Pen2, D-Pen5]enkephalin under physiological conditions is not substantially different to that measured in 50 mM Tris buffer using cell membrane fractions. Unlike DPDPE, the mu opioid agonists morphine, normorphine, PL-17, and DAMGO, have much lower affinity for the delta receptor measured under these conditions than is observed by studies using 50 mM Tris buffer. The results described here suggest that this assay may serve as a useful model of delta opioid receptor binding in vivo.

Topics: Animals; Binding, Competitive; Cell Survival; Endorphins; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalins; Hybrid Cells; Kinetics; Mice; Models, Biological; Morphine; Morphine Derivatives; Receptors, Opioid

In order to elucidate the substrate specificities for mu- and delta-opioid receptors, dimerization of the mu-specific morphine molecule was attempted, based on the hypothesis of the possible relationship between the molecular conformation of endogenous enkephalin and its selectivity for each opioid receptor. The NOR2 ([normorphine]N-CH2-CH2-N[normorphine]) thus synthesized exhibited agonist activity showing a preference for binding with the delta-opioid receptor, compared with the parent morphine molecule. Antagonist activity was also observed for NOR3 ([normorphine]N-CH2-CH2-CH2-N[normorphine]).

Topics: Animals; Binding, Competitive; Brain; Chemical Phenomena; Chemistry, Physical; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalins; In Vitro Techniques; Male; Mice; Morphine; Morphine Derivatives; Muscle Contraction; Muscle, Smooth; Protein Conformation; Rats; Rats, Inbred Strains; Receptors, Opioid; Receptors, Opioid, delta; Receptors, Opioid, kappa; Receptors, Opioid, mu; Vas Deferens

Intracellular recordings of membrane potassium current were made from rat locus coeruleus in vitro. The effects of agonists at mu-opioid receptors were studied on neurons from rats that had been chronically treated with morphine; these were compared with actions on neurons from control rats. Tolerance to the opioid-induced increase in potassium conductance was observed, and this was more pronounced for normorphine than for [Met5]enkephalin and [D-Ala2, Mephe4, Gly5-ol]enkephalin: experiments with the irreversible receptor blocker beta-chlornaltrexamine indicated that normorphine had lower intrinsic efficacy than [Met5]enkephalin and [D-Ala2 MePhe4, Gly5-ol]enkephalin. This adaptation was not due to any change of the properties of the potassium conductance activated by mu-receptors because both full and partial agonists at alpha 2-adrenoceptors, which couple to the same potassium conductance, were unchanged in their effectiveness; nor was it associated with any change in the affinity of mu-receptors for the antagonist naloxone. Naloxone had no effect on the neurons other than simple competitive reversal of the action of the mu-receptor agonists. These results demonstrate that 1) the mechanism responsible for tolerance in locus coeruleus neurons is specifically associated with mu-receptors and/or their coupling to potassium channels, 2) the intrinsic efficacy of an opioid determines the degree of tolerance observed, and 3) tolerance and physical dependence can be dissociated at the cellular level.

Topics: Animals; Drug Tolerance; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; In Vitro Techniques; Locus Coeruleus; Male; Membrane Potentials; Morphine; Morphine Derivatives; Naltrexone; Neurons; Potassium; Rats; Rats, Inbred Strains; Receptors, Opioid; Receptors, Opioid, mu

The irreversible non-selective opioid antagonist beta-chlornaltrexamine (beta-CNA) was used in combination with selective mu receptor protection by [D-Ala2, MePhe4, Gly(ol)5]enkephalin (DAGO) to produce an effective kappa receptor antagonism in the guinea-pig field-stimulated ileum preparation. Using a standard pre-treatment of 10(-7) M beta-CNA incubated for 15 min, DAG (10(-6)-10(-4) M) protected the response to the mu agonist normorphine while reducing the antagonism of the kappa agonist U50488 to a lesser extent. The concentration of DAGO which produced the most selective protection was 10(-5) M. This method was used to find the kappa selectivity of a series of opioid agonists. Of the compounds tested, butorphanol, dynorphin-(1-17), U50488, tifluadom, bremazocine and Mr 2034 were the most kappa-selective. The correlation with kappa agonist selectivity in vitro and effects in vitro on urine output in the rat is demonstrated.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Animals; Diuresis; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Guinea Pigs; Ileum; In Vitro Techniques; Male; Morphine Derivatives; Naltrexone; Pyrrolidines; Receptors, Opioid; Receptors, Opioid, kappa

Chronic treatment of rats with morphine caused tolerance to the membrane hyperpolarizing action of morphine in single locus coeruleus neurons in vitro. Tolerance was less pronounced for Try-D-Ala-Gly-MePhe-Gly-ol (DAGO) than for normorphine. No changes were found in affinity of naloxone for mu-receptors on these neurons, nor in the properties of the potassium conductance increase caused by mu-receptor activation. These results suggest that the mechanisms underlying tolerance involve either a reduction of the number of mu-receptors on each cell, or a reduced coupling of mu-receptors to potassium channels.

Topics: Animals; Drug Tolerance; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; In Vitro Techniques; Locus Coeruleus; Morphine; Morphine Derivatives; Neurons; Rats

Electrical stimulation (3 Hz, 2 msec duration, 5-12 V for 2 min every 20 min) of cortical slices from the rat, previously incubated with [3H]noradrenaline, evoked a release of tritium which was inhibited by morphine, normorphine, Tyr-D-Ala-Gly-MePhe-NH(CH2) 2OH ( RX783006 ) and D-Ala2-D-Leu5-enkephalin ( pIC30 5.90, 6.32, 7.45 and 6.74 respectively). Naloxone did not affect the release of tritium when given alone but antagonised the actions of the opioids, giving a Ke value of about 3 nM irrespective of the particular agonist used, which suggests an action at mu receptors. The delta opioid receptor blocker, ICI154129 , antagonised the opioids only in large concentrations (Ke 21300 nM). In slices previously incubated with [3H]5-hydroxytryptamine, electrical stimulation increased overflow of tritium but neither naloxone nor the opioid agonists affected evoked overflow of tritium at concentrations which were effective in slices incubated with [3H]noradrenaline. It is concluded that stimulation of mu opioid receptors may inhibit release of noradrenaline from central noradrenergic neurones and that these receptors are not present in significant numbers on neurones releasing 5-hydroxytryptamine in the cortex.

Topics: Animals; beta-Lipotropin; Cerebral Cortex; Clomipramine; Edetic Acid; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, Leucine; Enkephalin, Leucine-2-Alanine; Estradiol; In Vitro Techniques; Male; Morphine; Morphine Derivatives; Naloxone; Narcotics; Neurotransmitter Agents; Norepinephrine; Peptide Fragments; Rats; Rats, Inbred Strains; Receptors, Opioid; Serotonin