Application of receptor theory provides further evidence for the existence of the epsilon-opioid receptor in rat vas deferens.

In the present study, we analyzed the interaction of concomitantly-acting pairs of opioid agonists in inhibiting the electrically induced contraction of the rat vas deferens (RVD). The results when compared with the predictions of the law of mass action and the receptor theory (1,2) provide evidence for a specific receptor for beta-endorphin (beta-EP) in this organ. This receptor is not occupied by the delta-agonist D-Ala2-D-Leu5-enkephalin (DADLE), or the mu-agonists Sandoz FK-33824, fentanyl and etorphine in the range of concentrations necessary for these to exert their maximal agonistic action in the test.

[3H]Ethylketocyclazocine binding to mouse brain membranes: evidence for a kappa opioid receptor type.

The binding of the putative kappa agonist ethylketocyclazocine (EKC) to synaptosomal membranes of mouse brain was studied. This benzomorphan was able to bind to different opioid receptors. A portion of this binding was not inhibited by the agonist naloxone, even at high concentrations (10 microM).

Dynorphin B-29: chemical synthesis and pharmacological properties in opioid systems in vitro.

Porcine dynorphin B-29 was synthesized by solid phase procedures and purified using gel filtration, ion exchange, reverse phase liquid chromatography and preparative high pressure liquid chromatography (HPLC). Binding experiments using brain tissue indicated this peptide displaced mu and k ligands equally well and had significant, though less, affinity for delta. In isolated tissue systems, its potency ranked as follows: guinea pig ileum, mouse vas, rabbit vas, and rat vas.

Dynorphin A: inhibitory effect on other opiate ligand bindings in the mouse brain.

The opioid peptide dynorphin A antagonizes morphine-induced analgesia in vivo and inhibits opiate binding in vitro, although most of it is rapidly degraded under both conditions. The inhibitory effect was present even in tissue treated in vivo with dynorphin A and assayed in vitro without it. Shorter fragments of this peptide lacked these effects, indicating that the apparent potency did not result from a metabolite.

Dynorphin1-13: interaction with other opiate ligand bindings in vitro.

Dynorphin1-13 is a potent inhibitor of electrically-induced contractions in the guinea pig ileum, where it has the properties of kappa-(ethylketocyclazocine) type opioids. In the brain, however, it has no analgesic potency, yet inhibits that induced by morphine. To gain further insight into its mechanism of action in the latter system, we tested its ability to compete for the binding of several opiates to brain membranes in vitro.

[Leu5]enkephalin containing peptides derived from a common precursor: evaluation of opioid activity in in vitro bioassays.

Opioid peptides containing the sequence of [Leu5]enkephalin were studied in two isolated organ preparations sensitive to opiates, the guinea pig ileum (GPI) and the mouse was deferens (MVD). All peptides tested were able to inhibit the electrically stimulated contraction in both tissues by interacting with specific receptors sensitive to the antagonist naloxone. Some of these peptides, mainly the shorter sequences, showed considerable potency differences in the two systems, suggesting that at least two different types of receptors are involved.

Functional opiate receptor in mouse vas deferens: evidence for a complex interaction.

Treatment of the isolated mouse was deferens with the enzyme arylsulfatase (E.C. 3.

Long-term social isolation in the rat induces opposite changes in binding to alpha 1- and alpha 2-adrenoceptors in the brain and vas deferens.

Rats isolated at the time of calcification of the incisors show, after 14-18 months of social deprivation, an increased number of alpha 1-adrenoceptors labeled with [3H]WB 4101, both in the striatum and in the vas deferens, as well as a decreased number of alpha 2-adrenoceptors labeled with [3H]-clonidine in the vas deferens. Social isolation does not, however, modify the density of [3H]clonidine binding sites in the cerebral cortex. The functional state of alpha-adrenoreceptors in the vas deferens from isolated rats appears to correlate with the binding studies since the isolated tissue is hypersensitive to the contractile effect of exogenous noradrenaline and subsensitive to the inhibitory effect of clonidine on the electrically stimulated preparation.

Dynorphin-(1-13): the effect of in vivo treatment on opiate bindings in vitro.

Mice were injected intracerebroventricularly with different doses of dynorphin-(1-13) in vivo and decapitated after different intervals of time; crude P2 fractions were then prepared and used for in vitro binding with [3H]dihydromorphine (DHM) and [3H][D-Ala2,D-Leu5]enkephalin. Dynorphin pretreatment was found to decrease DHM binding in vitro in a both time-dependent and dose-dependent manner. Its maximal effect lasted from 30 min to 3 h and recovery took place in 6-12 h.

Differential modification of opiate receptor activity by arylsulfatase treatment.

Treatment with the enzyme arylsulfatase in vivo selectively attenuated the effect of analgesia induced by morphine, beta-endorphin or ethylketocyclazocine but not that induced by Sandoz FK33824 or D-ala2-D-leu5-enkephalin. The effect on morphine analgesia was indicated both by an increased morphine ED50 in the presence of a fixed dose of naloxone and by a decreased naloxone ED50 in the presence of a fixed dose of morphine. Arylsulfatase treatment in vivo also selectively affected in vitro ligand binding; Bmax values of the low affinity binding site of dihydromorphine, naloxone, D-ala2-D-leu5-enkephalin, D-ala2-met5-enkephalinamide and ethylketocyclazocine were decreased significantly while the Bmax values of the high affinity sites as well as the KD values of both the high and low affinity sites were affected little or not at all.

Endogenous opioid peptides: comparative evaluation of their receptor affinities in the mouse brain.

The affinities of certain endogenous opioid peptides and related sequences for mu, delta and k opiate receptors have been determined in membrane preparations from mouse brain. It was found that the KIs for the delta receptor changed very little when the sequence of the enkephalins was enlarged; on the contrary, the mu and especially the k activity were highly dependent not only on the specificity of the sequence but also on the length of the peptide. Most of the peptides have similar affinities for more than one receptor type.

Effect of human B-endorphin on the binding of different opiates to mouse brain membranes.

The competition of human B-endorphin (B-EP) for dihydromorphine (DHM) and D-Ala2-D-Leu5-enkephalin (DADLE) binding sites has been studied at three temperatures, 0 degree, 25 degrees, and 37 degrees C. B-EP is more effective in inhibiting mu and delta binding at 0 degree and 25 degrees than at 37 degrees C. This difference does not seem related to a temperature-dependent degradation of the peptide.

Dynorphin(1-10)amide: a potent and selective analog of dynorphin(1-13).

Dynorphin(1-10)amide was more potent than Dynorphin(1-13) in inhibiting the twitch of the mouse vas deferens (IC50 of Dynorphin(1-10)amide = 0.3 nM and IC50 of Dynorphin (1-13) = 4.0 nM).

Analgesic activity of substance P in rats: apparent mediation by met-enkephalin release.

The intracerebroventricular administration of Substance P (SP) produced a marked and short-lasting increase in the threshold for vocalization and vocalization afterdischarge in the rat after electrical stimulation of the tail. This effect was blocked by naloxone and potentiated by bacitracin, a peptidase inhibitor. The analgesic effect of SP was also blocked by the concomitant intraventricular injection of the specific antibody against the opioid peptide metenkephalin but not by the antibody against beta-endorphin.

Hyperactivity induced in rats by long-term isolation: further studies on a new animal model for the detection of antidepressants.

Male rats isolated at the time of calcification of the incisors show after 10 months of social deprivation a marked and sustained increase in locomotor activity together with other behavioural changes. The ages of separation as well as the prolonged isolation period appear to be critical with regard to the stability of the behavioural syndrome and to the sensitivity of the rats to different pharmacological treatments. The locomotor activity of solitary housed rats is selectively blocked after acute treatment with several antidepressants which are inactive according to the traditional laboratory procedures for detecting the activity of this class of drugs.