enkephalin--leucine-2-alanine and normorphine

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

The affinity of morphine, normorphine, methadone, Tyr-D-Ala-Gly-MePhe-NH(CH2)2(N-O)(CH3)2 (RX 783030), [D-Ala2,D-Leu5]enkephalin (DADLE), ketazocine and ethylketocyclazocine (EKC) were determined for their pharmacological receptors in two bioassay tissues, the guinea-pig ileum and the mouse vas deferens (MVD). The method involved the use of the irreversible antagonist, beta-chlornaltrexamine (beta-CNA), and the method of partial receptor blockade. The agonist concentration-effect curves were displaced to the right with decreasing maximum effect, a pattern typical of partial, irreversible blockade of receptors. The concentrations of beta-CNA required to produce a rightward displacement in the concentration-effect curves for different agonists, ranged between 2 and 3000 nM. No similarity was found between the IC50 and the dissociation constant (KA), values predicted to be equivalent only if a linear relationship exists between receptor occupation and observed effect; the dissociation constant for the agonists were between 3 and 218 times larger than the IC50 values. When methadone was used as the agonist in the guinea-pig ileum, beta-CNA produced parallel displacement of the concentration-effect curve, regardless of the blocking concentration chosen, preventing the determination of KA for this agonist, in this tissue; this problem was not encountered in the mouse vas deferens. The KA of morphine, RX 783030 and ketazocine were found not to differ in the guinea-pig ileum and mouse vas deferens. As expected, DADLE had significantly different affinity in the two tissues, showing 117-fold lower affinity in the guinea-pig ileum. Surprisingly, the normorphine affinity was found to be 7-fold higher in the guinea-pig ileum. While the difference in affinity of DADLE may be due to the suggested lack of functional delta receptors in the guinea-pig ileum, the difference in affinity seen with normorphine, but not morphine, in the two tissues is difficult to explain. Taken together with the insensitivity of methadone to beta-CNA blockade in the guinea-pig ileum, but not mouse vas deferens, the difference in the affinity of normorphine in these tissues may suggest the possibility of differences in local milieu of mu receptors or of mu receptor subtypes in the two tissues. The results provide fundamental information regarding opioid agonist affinity in two standard bioassays in vitro, and support the view of (1) a difference in receptors activated by DADLE in the guinea-pig

Topics: Animals; Cyclazocine; Enkephalin, Leucine; Enkephalin, Leucine-2-Alanine; Enkephalins; Ethylketocyclazocine; Guinea Pigs; Ileum; Male; Methadone; Mice; Morphine; Morphine Derivatives; Naltrexone; Receptors, Opioid; Vas Deferens

In the mouse isolated vas deferens the amplitude of excitatory junction potentials (e.j.p.s) recorded intracellularly from smooth muscle cells varied with the strength of stimulation. Receptor type selective opioids were tested in this preparation. The mu-agonist normorphine (2,000 nmol/l) reduced the amplitude of e.j.p.s and shifted the stimulus-response curve in a parallel way to the right. By contrast, the kappa-agonist U-50488 (1,000 nmol/l) and the delta-agonist [D-Ala2,D-Leu5]-enkephalin (2 nmol/l) caused a non-parallel shift of the curve. In addition, opioids having a lower selectivity for one type of receptor were also used. The preferential kappa-agonists ethylketocyclazocine (40 nmol/l) and dynorphin A1-13 (100 nmol/l) produced parallel and non-parallel shifts, respectively. Thus, normorphine and ethylketocyclazocine were more effective in depressing e.j.p.s evoked by low intensities of stimulation than those evoked by high intensities of stimulation. U-50488, dynorphin A1-13 and [D-Ala2,D-Leu5]-enkephalin caused an equal depression of e.j.p.s evoked by either intensity of stimulation. The preferential mu- and delta-antagonists naloxone (1,000 nmol/l) and ICI 154129 (10,000 nmol/l), reversed the action of the respective agonists normorphine and [D-Ala2, D-Leu5]-enkephalin. In addition, ICI 154129 (10,000 nmol/l) reversed the action of dynorphin A1-13, as well. The preferential kappa-antagonist MR-2266 (1,000 nmol/l) prevented the effect of both ethylketocyclazocine and U-50488. It is concluded that under the conditions of these experiments normorphine and ethylketocyclazocine acted at mu-, U-50488 at kappa-, and dynorphin A1-13 and [D-Ala2,D-Leu5]-enkephalin at delta-receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Analgesics; Animals; Cyclazocine; Dynorphins; Electric Stimulation; Enkephalin, Leucine; Enkephalin, Leucine-2-Alanine; Ethylketocyclazocine; In Vitro Techniques; Male; Mice; Morphine Derivatives; Muscle, Smooth; Neuromuscular Junction; Pyrrolidines; Receptors, Opioid; Receptors, Opioid, delta; Receptors, Opioid, kappa; Synaptic Transmission

The sigma opiates differ from other opiates in their stimulatory and psychotomimetic actions. The sigma opiate [3H](-)-SKF-10,047 has been used to characterize sigma receptors in rat nervous tissue. Binding of [3H](-)-SKF-10,047 to rat brain membranes was of high affinity, saturable, and reversible. Scatchard analysis revealed the apparent interaction of this drug with two distinct binding sites characterized by affinities of 0.03 and 75 nM (5 mM Tris-HCl buffer, pH 7.4, at 4 degrees C). Competition analyses involving rank order determinations for a series of opiates and other drugs indicate that the high-affinity binding site is the mu opiate receptor. The lower-affinity site (revealed after suppression of mu and delta receptor binding) has been identified as the sigma opiate/phencyclidine receptor. In vitro autoradiography has been used to visualize neuroanatomical patterns of receptors labeled using [3H](-)-SKF-10,047 in the presence of normorphine and [D-Ala2,D-Leu5]enkephalin to block mu and delta interactions, respectively. Labeling patterns differ markedly from those for mu, delta, or kappa receptors. The highest densities (determined by quantitative autoradiography) are found in the medial portion of the nucleus accumbens, amygdaloid nucleus, hippocampal formation, central gray, locus coeruleus, and the parabrachial nuclei. Receptors in these structures could account for the stimulatory, mood-altering, and analgesic properties of the sigma opiates. Although not the most selective sigma opiate ligand, [3H](-)-SKF-10,047 binds to sigma opiate receptors in brain, and this interaction can be readily distinguished from its interactions with other classes of brain opiate receptors.

Topics: Animals; Autoradiography; Binding, Competitive; Brain; Cell Membrane; Endorphins; Enkephalin, Leucine; Enkephalin, Leucine-2-Alanine; Male; Morphine Derivatives; Narcotics; Phenazocine; Rats; Rats, Inbred Strains; Receptors, Opioid; Tissue Distribution

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

The agonist potencies of normorphine and [D-Ala2,D-Leu5]enkephalin (DADL) have been determined on the mouse vas deferens preparation under different conditions. Reducing the temperature at which the assay was performed had little effect on the response to DADL whilst the Emax of normorphine was greatly reduced. Tissues rendered tolerant to either agent exhibited little, if any, cross-tolerance to the other drug. The results suggest the presence of at least two types of opiate receptor in the mouse vas deferens.

Topics: Animals; Enkephalin, Leucine; Enkephalin, Leucine-2-Alanine; In Vitro Techniques; Male; Mice; Morphine Derivatives; Muscle, Smooth; Receptors, Opioid; Temperature; Vas Deferens

Topics: Animals; Dose-Response Relationship, Drug; Enkephalin, Leucine-2-Alanine; Enkephalin, Methionine; Enkephalins; Guinea Pigs; Ileum; Morphine; Morphine Derivatives; Myenteric Plexus; Receptors, Opioid; Receptors, Opioid, delta

Intracellular recording were performed in vitro from dorsal root ganglion cell bodies isolated from adult rats. A- and C-type cells were distinguished according to the conduction velocity of their axons. Bath perfusion of normorphine, methionine-enkephalin (M-E) or [D-Ala2,D-Leu5]-enkephalin (DADL) did not alter membrane potential, threshold potential for spike initiation or the spike waveform either before or after addition of Ba2+ and 4-aminopyridine. Perfusion with gamma-aminobutyric acid (GABA) depolarized A- and C-type cells and decreased input resistance. Picrotoxin antagonized this action. Morphine or M-E applied by perfusion never altered the GABA-induced depolarizations. Iontophoretically administered morphine reduced the GABA-induced depolarization without alteration of resting membrane properties or spike form, while M-E increased the GABA-induced depolarization. In about 50% of cells tested M-E applied iontophoretically produced a reversible hyperpolarization associated with an elevated input resistance (120-200% control). None of the effects produced by the opioids were antagonized by naloxone applied by perfusion or iontophoretically. These results suggest that the cell bodies of spinal ganglion cells of adult animals lack opiate receptors.

Topics: Action Potentials; Aminopyridines; Animals; Barium; Enkephalin, Leucine-2-Alanine; Enkephalin, Methionine; Enkephalins; gamma-Aminobutyric Acid; Ganglia, Spinal; Iontophoresis; Morphine; Morphine Derivatives; Naloxone; Narcotics; Rats; Receptors, Opioid

Intracellular recordings were made from rabbit nodose ganglion cells in vitro. Morphine (up to 100 micro M), normorphine (up to 10 micro M) and D-Ala2, Leu5-enkephalin (DADLE) (up to 5 micro M) each had no detectable effect on the electrical properties of the cell membrane, except for local anesthetic-like actions at the highest concentrations which were not reversed by naloxone. Extracellular recordings were made from the infranodose vagus nerve in vitro using a sucrose gap method. No effects of morphine, normorphine or DADLE were detected on the resting potential, compound action potential or compound action potential enhanced by barium or tetraethylammonium. Moderate levels of stereospecific binding of tritiated dihydromorphine and DADLE were detected in both the nodose ganglion and vagus nerve. It is surmised that the radioligand binding sites on the nodose ganglion and vagus nerve are not functionally linked to detectable electrophysiological effects.

Topics: Action Potentials; Animals; Endorphins; Enkephalin, Leucine-2-Alanine; Enkephalins; Evoked Potentials; Kinetics; Morphine; Morphine Derivatives; Nodose Ganglion; Rabbits; Receptors, Opioid; Structure-Activity Relationship; Vagus Nerve

Low concentrations of the relatively selective opiate receptor agonists dihydromorphine and normorphine (mu receptor agonists) and D-Ala 2-D-Leu 5-enkephalin (a delta receptor agonist) were applied to single enteric neurons while the frequency of action potential firing was recorded. Most neurons that were inhibited by the mu agonists were also inhibited by the delta agonist, but the two receptors could be distinguished by the higher concentration of naloxone required to antagonize the delta agonist. The results indicate that enteric neurons bear both mu and delta receptors and that cell firing is inhibited if either receptor type is activated.

Topics: Animals; Dihydromorphine; Electric Conductivity; Endorphins; Enkephalin, Leucine-2-Alanine; Enkephalins; Guinea Pigs; Ileum; Morphine Derivatives; Myenteric Plexus; Neurons; Receptors, Opioid