Javier Garzón

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. The enkephalins are the most selective for the delta receptor. beta-Endorphin, BAM-12P, BAM-22P, peptide E and dynorphin A display the best potency at the mu site. Dynorphin A (1-8), dynorphin A and dynorphin B are the most selective for the k site.

Dynorphin(1-13), a long-lasting inhibitor of opiate receptor binding in vitro

The effect of dynorphin was determind on the binding of radioactive μ, δ and k ligands to mouse brain P2 fractions in vitro. The Bmax of high affinity sites of each ligand was lowered and the Kd was raised for k ligands. This suggests that the mode of interaction of dynorphin with brain is complex and may involve a site that allosterically affects the binding sites of other opiates.

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. Dynorphin1-13 inhibited the binding of all ligands examined, including dihydromorphine, D-Ala2-D-Leu5-enkephalin, ethylketacyclazocine (EKC) and naloxone. In all cases, it reduced the number of high affinity sites and, in the case of EKC, it also increased the Kd. We conclude that the mechanism of dynorphin inhibition is not simple rapidly reversible competition and is certainly not identical with respect to all opiate ligands.

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. The data suggest that the change induced by the enzyme may have been due to the alteration of certain constituents of the low affinity opiate binding site.

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). Binding assays indicated that two opioid peptides had similar profiles in that they enhanced dihydromorphine (DHM) binding in picomolar concentrations but displaced DHM binding in nanomolar concentrations (IC50 for Dynorphin(1-10)amide = 5 nM). In the mouse tail-flick assay, however, Dynorphin(1-10)amide showed a more selective action on morphine-induced analgesia. Although Dynorphin(1-10)amide had no significant analgesic activity by itself, it differed from the (1-13) analog by neither potentiating nor antagonizing morphine in naive animals. In tolerant animals, on the other hand, 50 microgram of this analog administered icv shifted the ED50 of morphine from 43.0(33.0-55.9) to 17.0 (12.4-23.3). Thus, Dynorphin(1-10)amide appears to be a more potent and selective analog of Dynorphin(1-13).

[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. Dynorphin-(1-17) displayed the highest agonistic potency in both preparations. In its case, there were no differences in IC50s nor in the shapes of the dose response curves in the two systems, suggesting a common receptor type; however, the reversal of its inhibitory effect following washout of the peptide was much more complete in the MVD than in GPI. Dynorphin B exhibited a higher potency in the GPI. Extension to dynorphin B-29 peptide did not induce changes in the agonistic activity in either system. An increase in one amino acid residue, dynorphin-(1-9) to -(1-10) or beta-neo-endorphin to alpha-neo-endorphin, resulted in a large potency increase in GPI and an opposite effect in MVD. While it has been reported that dynorphin interacts with the kappa opiate receptor in both mouse vas deferens and guinea pig ileum, our results suggest that the observable differences in the kinetics of the interaction in these systems could be due to the presence of different receptor types in these tissues.

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. Analysis of the Scatchard plots showed that dynorphin decreased the number of high affinity-binding site for DHM, while KD of this site was essentially unaltered. Neither Bmax nor KD of low affinity site were much affected. A similar decrease in high affinity Bmax for DADLE was also obtained; however, the recovery was even more rapid. Extra washes of tissue did not significantly increase the binding, but preincubation of tissue in the presence of morphine reversed the dynorphin effect and increased DHM binding significantly to almost control levels. The probable mechanism of dynorphin in decreasing opiate receptor binding is discussed.

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. Interestingly, all IC50s were reduced in the presence of peptidase inhibitors, particularly in the rabbit vas. These results indicate that dynorphin B-29 like dynorphin, interacts with multiple receptors in the brain, as well as in isolated tissue systems.

Mathematical modeling of opiate binding to mouse brain membrane

The heterogeneity of rat brain opiate receptors was examined by analyzing competition data. The binding of three prototypical tritiated opioid agonists, [3H]-dihydromorphine ([3H]-DHM), [3H]-D-ala2-D-leu5-enkephalin ([3H]-DADLE), and [3H]-ethylketocyclazocine ([3H]-EKC) was determined in the presence of varying concentrations of each of these unlabeled ligands, generating nine displacement curves. A computer program was then used to find the best fit of a model system to these data, assuming two, three or four independent binding sites. The best fit was a four-site model. One of these sites is specific for DHM; two are relatively selective for DHM and DADLE respectively, but also bind EKC. The remaining site binds only EKC with high affinity. These results, together with displacement data using naloxone, FK33824, and D-ala2-met5-enkephalinamide, are discussed in terms of current opiate receptor models.

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. DHM and DADLE high affinity binding sites are clearly distinguished by B-EP. The high affinity site for DHM and the low of DADLE are the more easily displaced by B-EP, but with different affinities. These sites are different in binding capacities, although distinct stoichiometric ratios of binding or the existence of isoreceptors could account for their identity.