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Biomedical subjects

A D Corbett

Publications and source records attributed to A D Corbett.

34 records · Page 2Linked to original sources
PubMed1985

10-Ketonaltrexone and 10-ketooxymorphone.

Ethylketocyclazocine (1) has greater kappa/mu selectivity than cyclazocine in brain binding assays. 10-Ketonaltrexone (11) and 10-ketooxymorphone (10) were prepared from naltrexone 3-methyl ether and oxycodone, respectively. Bioassays in the myenteric plexus longitudinal muscle preparation of the guinea pig ileum and in the mouse vas deferens, in addition to brain binding assays, demonstrated that 10 and 11 were far less potent than naltrexone (2) and oxymorphone (3) at mu sites and also had little affinity for kappa and delta sites. It is concluded that introduction of the 10-keto group in naltrexone and oxymorphone diminished opioid effects at all binding sites.

Animals↗
PubMed1985

Pre-incubation of guinea-pig myenteric plexus with beta-funaltrexamine: discrepancy between binding assays and bioassays.

The acute effects of beta-funaltrexamine and the effects of pre-incubation with this compound were examined in five in vitro assay tissues and in selective binding assays in homogenates of guinea-pig brain and myenteric plexus. In competitive displacement assays with selective ligands, beta-funaltrexamine had highest affinity for the mu-binding site in the myenteric plexus and brain of guinea-pig. Its affinity for the kappa-site was about 15% of that for the mu-site. Pre-incubation of the assay tissues with beta-funaltrexamine caused an increase in the IC50 values of mu- and delta-receptor agonists but not of kappa-agonists. Although in bioassays on the myenteric plexus-longitudinal muscle preparation of the guinea-pig, the IC50 value of the mu-receptor ligand [D-Ala2, MePhe4, Gly-ol5] enkephalin was increased up to 124 fold, its binding at the mu-site in homogenates of the preparation was not affected by this treatment. These findings indicate that the effects of pre-incubation with beta-funaltrexamine on agonist potency of the mu-receptor ligand are due to an interference with the coupling mechanism between the mu-binding site and the effector system.

Animals↗
PubMed1984

Hamster vas deferens contains delta-opioid receptors.

Electrical field stimulation of the hamster, isolated vas deferens produces regular contractions that are unaffected by opioid-receptor agonists which are selective for the mu- or kappa-receptor types. However, agonists which show a selectivity for the delta-opioid receptor produce dose-related inhibitions of the stimulation-evoked contractions. Response of delta-opioid receptor agonists are antagonized in a competitive fashion by the selective delta-receptor antagonist ICI 174864, and also by naloxone. The "kappa-opioid-agonist" benzomorphans, bremazocine and ethylketocyclazocine are antagonists in the hamster vas deferens; bremazocine is particularly potent in this regard. In conclusion, the hamster, isolated vas deferens may contain only delta-opioid receptors and provides a simple and specific test for the assay of activity at the delta-opioid receptor.

Animals↗
PubMed1984

Acute and persistent effects of beta-funaltrexamine on the binding and agonist potencies of opioids in the myenteric plexus of the guinea-pig ileum.

In binding assays with homogenates of the myenteric plexus-longitudinal muscle of the guinea-pig, beta-funaltrexamine is more potent at displacing mu-binding than kappa-binding. Incubation of homogenates with beta-funaltrexamine (100 or 1000 nM) for 30 min at 37 degrees C followed by repeated washing with drug-free Tris buffer does not alter the binding of either the selective mu-ligand [3H]-[D-Ala2, MePhe4,Gly-ol5]enkephalin or of [3H]-(-)-bremazocine made selective for the kappa-binding site by the addition of unlabelled mu- and delta-ligands. This observation is surprising since, after treatment with beta-funaltrexamine (100 nM), the IC50 values for the inhibition of the contraction of the myenteric plexus-longitudinal muscle by the mu-ligand [D-Ala2,MePhe4, Gly-ol5]enkephalin are increased 12-fold whereas the IC50 values obtained with the selective kappa-ligand U-50, 488H remain unaltered. It is proposed that the irreversible blockade produced by beta-funaltrexamine is not due to inhibition at the mu-site per se but to an interference with the link between the binding and the effector response.

Animals↗
PubMed1984

Selectivities of opioid peptide analogues as agonists and antagonists at the delta-receptor.

The endogenous opioid ligands interact with more than one of the mu-, delta- and kappa-binding sites. By the use of binding assays and bioassays, enkephalin analogues have been assessed for their selectivity for binding at the delta-binding site and for their agonist and antagonist activities at the delta-receptor. The electrically-induced contractions of myenteric plexus-longitudinal muscle preparations of the guinea-pig ileum were inhibited by mu- and kappa-receptor ligands. Inhibitions were seen with mu-, delta- and kappa-receptor ligands in the mouse vas deferens, mainly with mu-receptor ligands in the rat vas deferens and only with kappa-receptor ligands in the rabbit vas deferens. From observations on a considerable number of [Leu5] enkephalin analogues, it has been concluded that [D-Pen2, D-Pen5] enkephalin and [D-Pen2, L-Pen5] enkephalin are the most selective delta-agonists and that N,N-diallyl-Tyr-Aib-Aib-Phe-Leu-OH is the most selective antagonist (Aib = alpha-aminoisobutyric acid). The binding of these peptides at the delta-site is 99% of the total binding. As to potency, the agonists are superior to the antagonists.

Animals↗
PubMed1984

Radioligands for probing opioid receptors.

The three endogenous opioid precursors of almost 30000 Da are pro-opiocortin, proenkephalin and prodynorphin. Pro-opiocortin contains beta-endorphin, melanotropins and ACTH. Proenkephalin yields one [Leu5]enkephalin, three [Met5]enkephalins, one [Met5] enkephalyl-Arg-Arg-Val-NH2 (metorphamide or adrenorphin), one [Met5]enkephalyl-Arg-Gly-Leu and one [Met5]enkephalyl-Arg-Phe. [Leu5]enkephalin is common to all fragments of prodynorphin; its carboxyl extension by Arg-Lys leads to alpha- and beta-neo-endorphin and its carboxyl extension by Arg-Arg gives two dynorphins A and B of 17 and 13 amino acids, respectively. Another endogenous peptide is dynorphin A (1-8). The three main opioid binding sites are mu, delta and kappa. Their analysis has been facilitated by the synthesis of analogues of peptides and non-peptide compounds, which have selective agonist or antagonist action at only one site. The various physiological roles of the three types of the opiate receptor have so far not been sufficiently investigated.

3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cycloh↗
PubMed1983

Increase in potencies of opioid peptides after peptidase inhibition.

Various agents that have been reported to reduce the enzymatic degradation of the enkephalins have been tested for their ability to potentiate the activity of [Met5]enkephalin in three in vitro assay tissues. The greatest effect was obtained with the combination of bestatin (10 microM or 30 microM), captopril (10 microM), thiorphan (0.3 microM) and L-Leucyl-L-leucine (2 mM) which increased the potency of [Met5]enkephalin 18-fold in the guinea-pig myenteric plexus, 13-fold in the mouse vas deferens and 200-fold in the rat vas deferens. The increased potency is attributed to inhibition of the peptidases since the mixture of inhibitors did not change the activity of either normorphine or the metabolically stable synthetic opioid peptides. The potencies of the hexa-, hepta- and octapeptide C-terminus extensions of [Met5]enkephalin and [Leu5]enkephalin were increased by the peptidase inhibitors in all three preparations; the greatest effects were found in the rat vas deferens. No significant changes in the potencies of fragments of beta-endorphin longer than beta-endorphin-(1-19) were obtained. It may now be possible to inhibit enzymatic degradation of opioid peptides sufficiently to measure their release from neurones activated by electrical field stimulation.

Animals↗
PubMed1983

Characterization of opioid peptides in guinea-pig heart and skin.

Using reverse phase HPLC separations, and assay of eluate fractions in the mouse vas deferens, extracts of heart and skin from guinea-pigs were shown to contain several species of opioid-active material in low amounts (3-20 pmol/g as [Met5]enkephalin). Immunohistochemical studies revealed in the heart a small number of enkephalin-immunoreactive nerve fibres, particularly in cardiac ganglia and some small cells (paraganglionic cells, APUD cells). In the skin, enkephalin-immunoreactivity was confined to Merkel cells. Cardiac and cutaneous opioid peptides may modulate peripheral cardiovascular and sensory functions.

Animals↗
PubMed1983

Metorphamide: isolation, structure, and biologic activity of an amidated opioid octapeptide from bovine brain.

Acid acetone extracts of caudate nucleus from bovine brain were found to contain an amidated opioid octapeptide with the following structure: Tyr-Gly-Gly-Phe-Met-Arg-Arg-Val-NH2. The peptide has been named metorphamide. Bovine metorphamide appears to be derived by proteolytic cleavage from proenkephalin, the common precursor to [Met5]enkephalin and [Leu5]enkephalin. The cleavage within the precursor giving rise to the carboxyl terminus of metorphamide occurs at a single arginine residue and is followed by transformation of a carboxyl-terminal glycine into an amide group. Metorphamide was detected in bovine caudate nucleus extracts by radioimmunoassay, and it was purified to homogeneity by gel filtration and reversed-phase high performance liquid chromatography. Amino acid composition analysis and automated Edman degradation in the gas-phase sequencer confirmed the postulated amino acid sequence. Carboxyl-terminal amidation of bovine metorphamide was shown by stability to carboxypeptidase A digestion and full crossreactivity in a radioimmunoassay that required the carboxyl-terminal amide as part of the recognition site. A synthetic replicate of metorphamide as well as several synthetic analogs were tested for opioid activity in several bioassays and binding assays, and metorphamide was found to have a high mu-binding activity. Metorphamide is the only known naturally occurring opioid peptide that has a high mu-binding activity. The kappa-binding activity is approximately equal to 50% that of the mu-binding activity, but delta-binding activity is negligible.

Amino Acid Sequence↗
PubMed1981

Tetraethylammonium facilitates the stimulation-evoked loss of the enkephalins from the myenteric plexus of guinea-pig ileum.

The effects of electrical field stimulation on the contents of [Met]enkephalin and [Leu]enkephalin were determined in myenteric plexus-longitudinal muscle preparations of the guinea-pig small intestine. Cycloheximide (0.1 mM) was present in all experiments to prevent de nouveau biosynthesis. The two enkephalins were separated by high performance liquid chromatography and assayed on the mouse vas deferens. Stimulation with submaximal pulses (50 mA, 0.5 ms) at a frequency of 10 Hz caused maximal losses of about 35% of [Met]enkephalin and [Leu]enkephalin after 3 h (108000 pulses). The plot of log (enkephalin content) against number of pulses was steeper during the first 30 min than during the later periods. Tetraethylammonium bromide (TEA, 10 mM) increased the [Met]enkephalin and [Leu]enkephalin contents of the non-stimulated preparations by about 50%. When the preparations were stimulated in the presence of TEA at 50 mA and 1 Hz, the plots of loss of enkephalins against number of pulses were linear until the maximum of about 50% was reached. Compared with stimulation in the absence of TEA, the rate constant was 8 times greater for [Leu]enkephalin and 20 times greater for [Met]enkephalin. The absolute losses per pulse were about 13 times greater for [Leu]enkephalin and 27 times greater for [Met]enkephalin than in the absence of TEA. In the presence of bacitracin and a mixture of dipeptides, the enzymatic degradation of the enkephalins was sufficiently suppressed to cause an overflow of 30-60% of the enkephalins lost from their stores into the perifusing Krebs solution. Until it is possible to determine the preformed precursors, which are present in large quantities, the kinetics relationship between these precursors and the enkephalins cannot be investigated. A similar dilemma exists for the relationship between "released' enkephalins and the losses from their stores.

Animals↗
PubMed1980

Effects of electrical stimulation on the enkephalins in guinea-pig small intestine.

When myenteric plexus-longitudinal muscle preparations of the guinea-pig small intestine are stimulated electrically in the presence of cycloheximide there is a decrease in the content of enkephalins which is taken to be due to their release. With supramaximal stimuli, the release per pulse is similar at 1 and 10 Hz but it is lower with submaximal than with maximal stimuli. Met-enkephalin is more readily released than Leu-enkephalin.

Animals↗