Mouse Vas Deferens Preparations Research Articles

The use of μ opioid receptors in the mouse vas deferens for the study of opiate tolerance

We have previously shown that μ opioid receptors in the guinea pig ileum preparations are poor models for those in the central nervous system to study the phenomenon of opiate tolerance. Since we have shown subsequently that the μ opioid receptors in the mouse vas deferens preparation differ from those in the guinea pig ileum, we investigated the possibility that the μ receptors in the mouse vas deferens might be better models for the study of opiate tolerance. The degree of tolerance produced by incubation of mouse vas deferens preparations with morphine in vitro was relatively low. However, when mice were either implanted with morphine pellets (75 mg free base) or injected s.c. with 100, 50 or 25 mg/kg of morphine sulfate, a large magnitude of tolerance in their vasa deferentia was consistently observed. When pA 2 analyses were performed in the tolerant vasa deferentia (25 mg/kg) using naloxone, the pA 2 value was 7.55 ± 0.18 compared to the control value of 8.45 ± 0.24. Since the tissue became significantly more refractory to the effects of the opiate antagonist with the development of tolerance, which is the opposite of what happens with central μ receptors, we conclude that the μ opioid receptors in the mouse vas deferens are not adequate models for those in the central nervous system to study the phenomenon of tolerance.

Binaltorphimine-related bivalent ligands and their kappa opioid receptor antagonist selectivity.

In an effort to develop selective antagonists for kappa opioid receptors, bivalent ligands that contain opioid antagonist pharmacophores derived from naltrexone or other morphinans were synthesized and tested on the guinea pig ileum (GPI) and mouse vas deferens (MVD) preparations. The minimum requirements for kappa selectivity are at least one free phenolic OH group and one N-cyclopropyl or N-ally substituent. Several compounds (3, 8, 10) with kappa selectivity as good as or better than norbinaltorphimine (nor-BNI, 2) were discovered. The structure-activity relationship revealed that the pyrrole ring functions strictly as a spacer and does not contribute to kappa selectivity. The pharmacologic data suggest that only one antagonist pharmacophore may be required for kappa selectivity and that the other morphinan portion of the molecule confers selectivity by interacting with a unique subsite proximal to the antagonist pharmacophore recognition locus.

A non-opioid mechanism in the inhibitory effect of ginseng saponins on electrically evoked contractions of guinea-pig ileum and mouse vas deferens.

Both ginseng total saponins (GTS) and one of its constituents, protopanaxatriol saponins (PT), inhibited the electrically evoked contractions of guinea-pig ileum (GPI) in a concentration dependent manner in a range of 1-100 micrograms/ml, and this effect was irreversible at high concentrations of the saponins. Protopanaxadiol saponins (PD) had a transient and weak effect. On the other hand, in mouse vas deferens (MVD), the contractions were increased by PT and PD, however, GTS was almost without effect. The inhibitory effect of morphine was arithmetically increased by pretreatment with 100 micrograms/ml of these saponins in GPI preparations, while the inhibitory effect of the contractions was potentiated in MVD preparations. Neither the inhibition of contractions in the GPI preparation nor the facilitation of contractions in the MVD preparation by these ginseng saponins was reversed by 1 microM naloxone, in contrast to naloxone antagonism of morphine-induced contractions in both preparations. GTS and PT caused a dose-dependent inhibition of BaCl2-induced contraction of GPI. It is concluded that the mechanism on the inhibitory or facilitated effect of ginseng saponins on electrically evoked contractions in GPI and MVD preparations may be separated from the effect of opioids, and the mechanism may be based on the direct action of the saponins on smooth muscles preparations.

Opioid agonists and antagonists. Peptides containing N-terminal allyl groups and/or a thiomethylene linkage in place of a peptide bond

Peptides containing N-allyl or N,N-diallyl groups at the N-terminus have been synthesized as potential opioid antagonists. A number of analogues with an amide bond replaced by a thiomethylene group have also been prepared. In brain binding assays and in guinea-pig ileum and mouse vas deferens preparations, the analogues generally displayed low affinity for µ- and δ-receptors as well as agonist activities in in vitro tests. N,N-Diallyl-Phe-D-Ala-Phe-Gly-NH2(28) was found to be a moderately potent but highly selective antagonist at δ opiate receptors.

Distinctive effect of ginseng saponins on development of morphine tolerance in guinea-pig ileum and mouse vas deferens.

Studies on the effect of ginseng saponins on the development of tolerance to morphine have been carried out using isolated preparations of guinea-pig ileum (GPI) and mouse vas deferens (MVD). Incubation of GPI preparation with morphine resulted in the development of tolerance to the inhibitory effect of morphine on the electrically evoked contractions. Ginseng total saponins and one of the constituents, protopanaxatriol saponin, suppressed the development of morphine tolerance in a concentration dependent manner in GPI preparation, though another constituent, protopanaxadiol saponin, did not affect the tolerance development substantially. In the MVD preparation, the development of tolerance to the morphine effect was observed as well, but none of the ginseng saponins affected it. It has been well established that electrically evoked contractions of GPI and MVD are mediated by acetylcholine and norepinephrine, respectively, and presumably their release is regulated presynaptically by opioid receptors. The fact that ginseng saponins suppressed the development of morphine tolerance only in the GPI preparation suggest that the inhibitory effect is mediated through and effect on the cholinergic system, without the involvement of direct action on opioid receptors.

Partial characterization of a novel endogenous opioid in human cerebrospinal fluid

Human cerebrospinal fluid (CSF) contains many uncharacterized endogenous opioids, in addition to the known enkephalins, endorphins, and dynorphins. These opioids may be separated by gel filtration chromatography and identified by radioreceptor assay for opioid activity. One region of the chromatographic elution profile, designated “Peak B” has previously been shown to be related to the pain status of chronic pain patients. We now report that human Peak B isolated from the CSF of pain-free elective surgery patients is present at a typical concentration equivalent in activity to 1.4 pmol of morphine sulfate per ml of CSF measured by radioreceptor assay. At a dose of 0.06 and 0.12 pmol morphine sulfate equivalents of CSF (MSE), injected into the cerebroventricular system of the mouse, Peak B produced an antinociceptive effect, the intensity and duration of which was dose-dependent and which was antagonized by naloxone. The mouse vas deferens (MVD) preparation was inhibited by Peak B in a manner that was sensitive to antagonism by naloxone only at low (< 1.0 μM) but not at higher (>6.0 μM) concentrations of the antagonist. Peak B activity in the MVD assay was unaffected by treatment with trypsin or α-chymotrypsin.

Synthesis and biological activity of some glucose-enkephalin conjugates.

Two O-glucopeptides, H-Tyr(beta-D-Glc)-Gly-Gly-Phe-OH(10) and H-Tyr(beta-D-Glc)-Gly-Gly-Phe-Leu-OH (11), having the amino acid sequence of enkephalin, were synthesized to determine the influence of the carbohydrate molecule on the biological activity and conformation of these opioid peptides. The synthesis were carried out in a stepwise and/or direct manner by fusing the activated O-glucosylpseudourea intermediate with suitably protected amino acid or peptide derivatives, followed by hydrogenolytic removal of protecting groups. The pure compounds were tested for opiate-like activity by using the guinea pig ileum (GPI) and mouse vas deferens (MVD) preparations.

ChemInform Abstract: Synthesis and Biological Activity of Analogues of β‐Chlornaltrexamine and β‐Funaltrexamine at Opioid Receptors.

AbstractThe bromoacetamide (I) reacts with diethanolamine (II) to yield (III) which is converted to the bischloroethylamino derivative (IV).

ChemInform Abstract: Investigation of 4‐(3‐Hydroxyphenyl)‐4‐methylpipecolic Acid as a Conformationally Restricted Mimic of the Tyrosyl Residue of Leucine‐Enkephalinamide.

Analogues of leucine-enkephalinamide containing N-terminal cis- or trans-4-(3-hydroxyphenyl)-4-methylpipecolic acid were prepared to examine the conformational requirements of the N-terminal tyrosyl residue in opioid activity. The diastereomeric amino acids were prepared and purified by semipreparative HPLC before incorporation into the peptide. Spectroscopic analysis based on proton nuclear Overhauser enhanced differential spectroscopy (NOEDS) allowed assignment of the cis and trans stereochemistry. Despite spatial analogy between the trans isomer 5 and leucine-enkephalinamide, it possessed neither opioid agonist nor antagonist activity in the guinea pig ileal longitudinal muscle (GPI) or mouse vas deferens (MVD) preparations. Possible explanations for this inactivity are discussed.

Probes for narcotic receptor mediated phenomena. 13. Potential irreversible narcotic antagonist-based ligands derived from 6,14-endo-ethenotetrahydronororipavine with 7-(methoxyfumaroyl)amino, (bromoacetyl)amino, or isothiocyanate electrophiles: chemistry, biochemistry, and pharmacology.

N-Allyl-, N-(cyclopropylmethyl)-, and N-propyl-endo-ethenotetrahydronororipavines (N-substituted 6,14-endo-etheno-4,5-epoxy-3-hydroxy-6-methoxymorphinans) were synthesized with potential acylating or alkylating moieties at the C-7 position (isothiocyanato, (bromoacetyl)amino, and (methoxyfumaroyl)amino) and examined in vivo for their narcotic agonist and antagonist activities and for their ability to interact with opioid receptors in vitro. The N-(cyclopropylmethyl)-substituted compounds were found to have the highest affinity for opioid receptors among these N-substituted compounds, although all of them were found to be reasonably potent narcotic antagonists in the mouse tail flick vs. morphine assay. Their in vivo potency ranged from 1/8 to 4 times that of nalorphine on intravenous injection in mice. Rat brain membrane binding studies indicated that the compounds interacted with opioid receptors with potencies that ranged from 0.5 times that of morphine (8c, 9c, and 10c) to 0.017 that of morphine (8b). Among the compounds studied here, only the previously reported isothiocyanato compound (10c) and (methoxyfumaroyl)amino compound (8c) interacted irreversibly and selectively with mu or delta opioid receptors, respectively, in assays using NG108-15 neuroblastoma-glioma hybrid cells and/or in a rat brain membrane preparation. Both 8c and 10c were found to interact irreversibly, to a limited extent, with kappa opioid sites in rat brain membranes in which the mu and delta opioid receptors were depleted by interaction with the mu-selective irreversible ligand BIT and the delta-selective irreversible ligand FIT. Neither compound showed irreversible actions in the electrically stimulated mouse vas deferens preparation.

Synthesis and biological activity of analogues of beta-chlornaltrexamine and beta-funaltrexamine at opioid receptors.

beta-Chlornaltrexamine and beta-funaltrexamine analogues 4-7 with different length "arms" to which an electrophilic moiety is attached were synthesized in an effort to obtain affinity labels that would selectively and irreversibly block specific opioid receptor types and subtypes. One of the compounds, 4, was a potent, irreversible blocker of opioid receptors in the guinea pig ileum and mouse vas deferens preparations. The results of this study suggest that nucleophiles that are remote from the recognition locus are capable of alkylation by reactive electrophiles.

Investigation of 4-(3-hydroxyphenyl)-4-methylpipecolic acid as a conformationally restricted mimic of the tyrosyl residue of leucine-enkephalinamide.

Analogues of leucine-enkephalinamide containing N-terminal cis- or trans-4-(3-hydroxyphenyl)-4-methylpipecolic acid were prepared to examine the conformational requirements of the N-terminal tyrosyl residue in opioid activity. The diastereomeric amino acids were prepared and purified by semipreparative HPLC before incorporation into the peptide. Spectroscopic analysis based on proton nuclear Overhauser enhanced differential spectroscopy (NOEDS) allowed assignment of the cis and trans stereochemistry. Despite spatial analogy between the trans isomer 5 and leucine-enkephalinamide, it possessed neither opioid agonist nor antagonist activity in the guinea pig ileal longitudinal muscle (GPI) or mouse vas deferens (MVD) preparations. Possible explanations for this inactivity are discussed.

Irreversible blockage of opioid receptor types by ester homologues of beta-funaltrexamine.

A series of ester homologues 2-5 of the mu receptor nonequilibrium antagonist beta-funaltrexamine (1, beta-FNA) was synthesized. These ligands were of interest in our investigation of the relationship between the structure of the ester function and the ability to irreversibly block mu opioid receptors. While all of the ligands were potent reversible agonists in the guinea pig ileum (GPI) and mouse vas deferens (MVD) preparations, most appeared to behave as irreversible antagonists of morphine. The benzyl 5 and phenethyl 6 esters possessed irreversible mu antagonist potency that was of similar magnitude to that of beta-FNA in the GPI. In the MVD, all esters appeared to irreversibly block the agonist effect of morphine, but none of the compounds irreversibly antagonized [D-Ala2,D-Leu5]enkephalin to a significant degree. [3H]Dihydromorphine displacement studies revealed no relationship between the affinity of the esters 1-6 and the irreversible blockage of mu receptors in the GPI or MVD. Possible reasons for the observed structure-activity relationship are discussed.

The contribution of intrinsic activity to the action of opioids in vitro.

The effects of opioids were compared in five field-stimulated isolated tissue models, the guinea-pig ileum and vasa deferentia from rat, rabbit and mice of the Alderley Park and C57BL/6 strains. Although the mu-receptor agonist [D-Ala2, MePhe4, Gly-ol5] enkephalin appeared to act at similar receptors in the guinea-pig ileum, rat vas deferens, mouse vas deferens and C57BL/6 mouse vas deferens preparations, its potency varied considerably between these preparations. Similar potency differences were also observed with the kappa-agonist, ethylketocyclazocine. It is proposed that these variations in potency reflect differences in the number of spare receptors present in each model. The finding that some drugs which have agonist activity in the more sensitive preparations behave as antagonists in the less sensitive tissues supports this proposal and highlights the importance of intrinsic activity in determining the action of opioids. Many of the prototypic opioid agonists were found to be either partial agonists (eg. morphine and bremazocine) or to possess affinity for more than one receptor type (eg. ethylketocyclazocine, Mr 2034).

Purification and characterization of an opioid antagonist from a peptic digest of bovine .KAPPA.-casein.

A chloroform/methanol extract of a peptic digest of bovine K-casein had binding activity to opioid receptors of rat brain. The active peptide was isolated by reverse-phase liquid chromatography on ODS and CN columns. The structure of the peptide was Ser-Arg-Tyr-Pro-Ser-Tyr-OCH3, which corresponds to the methyl ester of the 33 - 38th residues of κ-casein. The peptide was esterified during the extraction process, and the structure was confirmed by a chemical synthesis. The IC50 value of the peptide in a radioreceptor assay in the presence of 1 HM [3H]naloxone was 15 μM. The peptide did not have opioid agonistic activities in any of the assay systems tested. In the guinea pig ileum and rabbit vas deferens preparations, however, the peptide antagonized with morphiceptin and dynorphin A-(1-13), respectively. The peptide did not antagonize with [Leu]enkephalin in the mouse vas deferens preparations. Therefore, the peptide was an antagonist selective for the μ- and κ-types of opioid receptors. The peptide was named casoxin.

ChemInform Abstract: SYNTHESIS AND BIOLOGICAL EVALUATION OF PHOSPHONAMIDATE PEPTIDE INHIBITORS OF ENKEPHALINASE AND ANGIOTENSIN‐CONVERTING ENZYME

AbstractDie Verbindungen (I) werden synthetisiert und auf Inhibierung von Enkephalinase und ACE (angiotensin‐converting enzyme) untersucht.

Determination of the receptor selectivity of opioid agonists in the guinea-pig ileum and mouse vas deferens by use of beta-funaltrexamine.

The irreversible inhibitor of mu-opioid receptor-mediated effects, beta-funaltrexamine (beta-FNA), was used to investigate the selectivity of various opioid agonists at mu-opioid receptors in the electrically stimulated guinea-pig ileum and mouse vas deferens preparations in vitro. In the guinea-pig ileum, pretreatment with beta-FNA (3 X 10(-8) - 3 X 10(-6)M) produced a concentration-dependent antagonism of the inhibitory effect produced by the mu-opioid receptor agonist [D-Ala2, MePhe4, Gly(ol)5]enkephalin (DAGO). High concentrations of beta-FNA (3 X 10(-6) - 1 X 10(-5)M) also antagonized the inhibitory effects of the kappa-opioid agonist U50488. Pretreatment of guinea-pig ileum with beta-FNA at 1 X 10(-6)M resulted in blockade of the effect of some opioid agonists. The compounds which showed the largest rightward shifts in their concentration-response curves, and hence the greatest mu/kappa opioid receptor selectivity, were nalbuphine, [D-Ser2, Leu5]enkephalinyl-Thr6(DSLET), morphine, DAGO and normorphine. Responses to tifluadom, Mr 2034, ethylketocyclazocine, butorphanol, nalorphine, proxorphan and U50488 were not inhibited by beta-FNA. In the mouse vas deferens, pre-treatment with beta-FNA (1 X 10(-6)M) produced a similar shift in the dose-response curves for normorphine as in the guinea-pig ileum. The concentration-response curves for the delta-receptor agonists [D-Ala2, D-Leu5] enkephalin (DADLE) and DSLET were, however, also shifted, indicating that beta-FNA will also block delta-opioid receptors. Since beta-FNA does not block kappa-opioid receptor-mediated effects, it can be used in the guinea-pig ileum preparation as a selective mu-receptor inhibitor. However, its lack of selectivity between mu- and delta-opioid receptors should be taken into account in many other isolated tissues and experiments in vivo.

TENA, a selective kappa opioid receptor antagonist

A number of opioid antagonists (TENA, naloxone, Mr 2266, WIN 44441) were evaluated for their selectivity in antagonizing the effect of mu, kappa, and delta agonists in the guinea pig ileum (GPI) and mouse vas deferens (MVD) preparations. Among these four antagonists, TENA was the most potent and the only ligand which was selective for kappa receptors. In this regard TENA was approximately 27-times more effective in antagonizing kappa agonist, U-50488H, relative to the mu agonist, morphine, and it was about 5-times more effective against ethylketazocine (EK) relative to morphine. At the same concentration (20)nM) TENA did not significantly antagonize the delta agonist, [D-Ala 2, D-Ala 5]enkephalin (DADLE), in the MVD. Also, TENA was more effective than naloxone, EK, or U-50488H in protecting kappa receptors study indicate that TENA is the most selective kappa antagonist yet reported.

Comparison of “selective” opiate receptor antagonists on the isolated mouse vas deferens

The selectivity and relative potencics of opiate receptor antagonists were compared on the mouse vas deferens preparation. ICI-174864 was found to be a highly selective antagonist at delta opiate receptors equal in potency to naltrexone in blocking the actions of delta agonists. Although less potent than naltrexone, beta-funal trexamine (beta-FNA) and Mr-1452, like naltrexone, were less selective in that they blocked the actions of mu, delta and kappa agonists. The relative potencies of beta-FNA and Mr-1452 in antagonizing the three types of agonists also were similar to naltrexone.

Morphine, but not diacetyl morphine (heroin), possesses opiate antagonist activity in the mouse vas deferens

In an attempt to determine whether or not it is possible to differentiate between the efficacy of μ and δ agonists, activities of morphine, nomorphine, diamorphine, methionine enkephalin (ME) and the μ selective peptides RX 783006 (D-Ala 2 MePhe 4 Gly (ol) 5 - Enkephalin) and RX 783016 (Tyr -D-AlaGlyMePheNH(CH 2) 2 NMe 2) have been investigated in mouse vas deferens preparations stimulated by trains of high frequency (10 Hz) pulses. In the present study both the μ and δ selective peptides were found to possess equal efficacy (producing 80–90% inhibition of the contractions of the MVD). However, morphine which is accepted as the prototype pure μ agonist, was found to possess very low efficacy (reducing contractions by < 30%) and to act as an antagonist of both μ and δ peptides, being 35–50 times more effective against the μ agonists. The antagonist potency of morphine was approximately 1% that of naloxone at the μ receptor and 0.5% that of naloxone at the δ receptor. Diamorphine, in contrast to morphine, appears to be a relatively pure agonist in this preparation.