The mu-opioid activity of kappa-opioid receptor agonist compounds in the guinea pig ileum.

On the basis of their in vivo activity and binding affinity, nalorphine and (-)SKF 10,047 were classified as mixed agonist/antagonist compounds. However, in isolated tissue preparations without a selective antagonist to block their agonist effect, the characterization of these compounds and the determination of their antagonist activity were very difficult. Nor-binaltorphimine, a selective kappa-opioid receptor antagonist, was used in the longitudinal muscle preparations of the guinea pig ileum to block the kappa-agonist activity of nalorphine and (-)SKF 10,047. In the absence of their kappa-agonist activity, we were able to determine the mu-antagonist activity using the mu-selective agonist DAMGO ([D-Ala2,N-Me-Phe4,Gly-ol5]enkephalin). The pA2 values for nalorphine and (-)SKF 10,047 were 7.50 and 7.69, respectively.

Determination of activity for nociceptin in the mouse vas deferens.

The recently discovered neuropeptide nociceptin was found to inhibit electrically induced contractions of the mouse vas deferens. Nociceptin and its 14-Tyr analog were each partial agonists, but with high affinity (ED50 of 20 nM). This activity was not opioid in nature, as it was not inhibited by either selective or non-selective opiate antagonists.

The in vitro pharmacological characterization of naloxone benzoylhydrazone.

On the basis of its in vivo activity and binding affinity, naloxone benzoylhydrazone has been characterized as a kappa 3-opioid receptor agonist and a mu-opioid receptor antagonist. This paper continues its pharmacological characterization with the help of isolated tissue preparations. Naloxone benzoylhydrazone was found to have partial agonist activity in the guinea pig ileum longitudinal muscle/myenteric plexus preparation. As an antagonist, naloxone benzoylhydrazone is similar to naloxone, with pA2 values of 8.8, 7.8, and 7.8 for mu-, delta-, and kappa 1-opioid receptors, respectively. Its agonist activity in the guinea pig ileum preparation was not influenced by beta-funaltrexamine treatment but was reversed by the selective kappa-opioid receptor antagonist nor-binaltorphimine and by the irreversible kappa 1-opioid receptor blocker UPHIT (1S,2S)-trans-2-isothiocyanato-4,5-dichloro-N-methyl-N-[2-(1- pyrrolidinyl)-cyclohexyl] benzeneacetamide. The presence of kappa 3-opioid receptors could not be demonstrated by [3H]naloxone benzoylhydrazone binding in the guinea pig ileum longitudinal muscle/myenteric plexus preparation. From these studies it is concluded that the partial agonist activity of naloxone benzoylhydrazone in this bioassay is probably due to the activation of the kappa 1-opioid receptors.

Comparison of kappa 2-opioid receptors in guinea pig brain and guinea pig ileum membranes.

The presence of kappa-opioid receptor subtypes has been clearly established in guinea pig brain. Using [3H]bremazocine in the presence of reversible blockers of mu, delta and kappa 1 receptors, two additional binding sites can be determined in guinea pig brain membranes. The site with higher affinity for the opioid ligands represents kappa 2, while the other site has low affinity and is poorly characterized. The kappa 2 site has high affinity for ethylketocyclazocine and other benzomorphans, as well as for the dynorphin gene products tested. The dynorphin analogs have no appreciable affinity for the low affinity site, so this site should not be called a kappa receptor. With an appropriate membrane preparation, kappa 2 binding can also be demonstrated in the guinea pig ileum. Binding affinities for selected ligands at kappa 2 in guinea pig ileum membranes are very similar to affinities found in brain membranes.

A guinea pig ileum preparation devoid of functional kappa receptors: a new in vitro pharmacologic assay for mu-opioid ligands.

Guinea pig ileum longitudinal muscle/myenteric plexus preparations contain functional mu- and kappa-opioid receptors. A preparation with blocked kappa receptors was obtained by pretreating guinea pigs with 0.1-10.0 mg/kg intraperitoneal doses of a new kappa-selective affinity label, DIPPA (2-(3,4-dichlorophenyl)-N-methyl-N-[1S-1-(-3-isothiocyanatophenyl)-2-(-1 -pyrrolidinyl) ethyl] acetamide). Determination of IC50 values for the mu-selective agonist DAMGO ([D-Ala2,MePhe4,Gly(ol)5]-enkephalin) and the kappa-selective agonist U 69,593 ([5alpha,7alpha,8beta]-(+)-N-methyl-N-[7-(1-pyrrolidin yl)-1-oxaspiro-(4,5)-dec-8-yl]) showed that 0.5 mg/kg DIPPA at 48 h produced reliable, near-complete blockade of kappa-opioid activity but a minimal shift for kappa-opioid agonism. This new assay should be useful for studying mixed agonists/antagonists that produce strong kappa-opioid receptor agonism, which prevents determination of mu-opioid receptor antagonism.

Binding, pharmacological and immunological profiles of the delta-selective opioid receptor antagonist HS 378.

HS 378 is a recently developed indolomorphinan with high selectivity and antagonist potency at the delta-opioid receptor. The present study was performed to characterize the opioid binding properties and pharmacological and immunological activity of HS 378 and to compare them with those of two well-known delta-opioid receptor antagonists, naltrindole (NTI) and naltriben (NTB). In vitro opioid receptor binding profiles were determined in rat brain homogenates. HS 378 showed 4.7- and 2.4-fold higher mu/delta selectivity compared to NTI and NTB, respectively. In the [35S]GTPgammaS functional assay carried out in cell lines expressing cloned human opioid receptors, HS 378 was found to be a pure delta-opioid receptor antagonist. In vitro, exposure of HS 378 resulted in an apparent dose-related suppression of concanavalin A induced rat T-lymphocyte proliferation with an IC50 value of 0.54 microM. NTI showed also immunosuppression with an IC50 value of 6.93 microM, whereas NTB had no effect. The IC50 of HS 378 was 13 times lower than that of NTI and 8 times higher than that of cyclosporin A. Taken together, our findings indicate that the small molecule HS 378 has properties that may be of therapeutic value in the setting of human inflammatory diseases.

Binding and in vitro activities of peptides with high affinity for the nociceptin/orphanin FQ receptor, ORL1.

Fifteen hexapeptides having high affinity for the opioid-like receptor ORL1 were identified from a combinatorial library containing more than 52 million different hexapeptides. The five compounds with the highest affinity were characterized further by use of a variety of in vitro models. Binding studies indicated that these five peptides have affinity for ORL1 in the nanomolar range, similar to the recently discovered endogenous ligand called nociceptin and orphanin FQ (N/OFQ). The activity of these compounds was investigated in three different assays: stimulation of [35S]GTPgammaS binding and inhibition of forskolin-stimulated cAMP accumulation in Chinese hamster ovary cells transfected with ORL1, and inhibition of electrically induced contractions in the mouse vas deferens. In each assay, the five hexapeptides acted as partial agonists. The EC50 values for stimulation of [35S]GTPgammaS binding and inhibition of cAMP accumulation were in the range of that for N/OFQ, but maximal effects ranged from 70 to 90% of N/OFQ in the cAMP assay, and 30 to 60% of N/OFQ in the GTPgammaS assay. The positive hexapeptides identified were found to have minimal structural similarity to N/OFQ. The peptides are positively charged, which could enable them to bind to the negatively charged second extracellular loop thought to be a likely binding site for N/OFQ.