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Biomedical subjects

H W Kosterlitz

Publications and source records attributed to H W Kosterlitz.

At least 55 records · Page 3Linked to original sources
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

Interactions of some 4-anilinopiperidines and 4-phenylpiperidines with the mu, delta- and kappa-binding sites.

The binding and pharmacological profiles of some 4-anilinopiperidine and 4-phenylpiperidine analogues were determined. The potency of the compounds to displace the binding of selective tritiated ligands was measured in homogenates of guinea-pig brain at 25 degrees C. All the compounds tested had high affinity for the mu-binding site, low affinity for the delta-site and were almost inactive at the kappa-site. The compounds were also tested for agonist activity in the guinea-pig ileum and the vasa deferentia of the mouse and rat. The pharmacological and binding profiles were compared.

Animals↗
PubMed1983

[3H]-dynorphin A (1-9): binding characteristics and degradation profile in brain homogenates.

[3H]-Dynorphin A (1-9) is rapidly degraded in brain homogenates, even at 0 degree C. Although protection from degradation at O degree C, and to a lesser extent at 25 degrees C, was obtained with a combination of bestatin, L-leucyl-L-leucine, L-arginyl-L-arginine and captopril, these peptidase inhibitors had little effect on binding at 0 degree C. At the kappa-site, the maximum binding capacity of [3H]-dynorphin A (1-9) at 0 degree C was similar to that at 25 degrees C but it is suggested that, even after suppression of mu- and delta-binding, [3H]-dynorphin A (1-9) may bind to more than one site for which it has differing affinities at 0 degree C.

Animals↗
PubMed1983

Lysine substitution increases mu-selectivity of potent di- and tri-peptide enkephalin analogs.

A general trend toward increased mu-selectivity has been reported in two classes of minimal structure enkephalin analogs which contain the essential structural and functional information for potent enkephalin-like biological activity. A single Lys residue added to the amino terminal of several tripeptide amides, and to the potent dipeptide amide Tyr-DAla-phenylpropylamide, causes a further increase in mu-selectivity as determined in the stimulated guinea pig ileum (GPI) and mouse vas deferens (MVD) assays, and in receptor binding studies in homogenates of guinea pig brain. The effect of Lys substitution was a significant decrease in the delta-related MVD potencies.

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↗
PubMed1982

Interaction of p-nitrophenylalanine enkephalins with mu-, delta- and kappa-subtypes of the opiate receptor.

Substitution of L-p-nitrophenylalanine for phenylalanine in position 4 of [D-Ala2, KLeu-NH2(5)]enkephalin analogues increased their affinity for mu-, delta- and kappa-binding sites in guinea-pig brain, increased their potency to inhibit electrically evoked contractions of the guinea-pig ileum and mouse vas deferens, and increased their analgesic potency in the mouse tail flick test, Introduction of L-adamantylalanine, but not of L-neopentylglycine, in position 5 resulted in a loss of activity.

Animals↗
PubMed1982

Spectrum of the mu, delta- and kappa-binding sites in homogenates of rat brain.

1 In homogenates of rat brain, the binding characteristics of tritiated opiates and opioid peptides were examined and the relative capacities of mu-, delta- and kappa-binding sites of the opiate receptor determined by saturation analysis.2 In competition experiments, binding of the selective mu-ligand [(3)H]-[D-Ala(2),MePhe(4),Gly-ol(5)]enkephalin at the mu-site was displaced by [D-Ala(2),D-Leu(5)]enkephalin with rather low affinity (K(I) = 12.6 nM) and more readily by the ketazocine-like compounds (-)-ethylketazocine (K(I) = 3.1 nM) and (-)-bremazocine (K(I) = 0.32 nM), which also displaced the binding of [(3)H]-[D-Ala(2),D-Leu(5)]enkephalin from the delta-site. In contrast, the binding to the kappa-site was easily displaced by ethylketazocine (1.0 nM) and bremazocine (0.37 nM) but not by the mu-ligand [D-Ala(2),MePhe(4),Gly-ol(5)]enkephalin (K(I) = 2000-3000 nM) or the delta-ligand [D-Ala(2),D-Leu(5)]enkephalin (K(I) > 20,000 nM).3 The dissociation equilibrium constant (K(D)) and the binding capacity (pmol/g) of the mu-binding site were determined with the selective mu-ligand [(3)H]-[D-Ala(2),MePhe(4),Gly-ol(5)]enkephalin. For the delta-site, [(3)H]-[D-Ala(2),D-Leu(5)]enkephalin was used in the presence of unlabelled [D-Ala(2),MePhe(4),Gly-ol(5)]enkephalin in order to suppress cross-reactivity to the mu-binding site. For the estimation of kappa-binding, [(3)H]-(+/-)-ethylketazocine or [(3)H]-(-)-bremazocine were used in the presence of unlabelled mu- and delta-ligands for the suppression of cross-reactivities to the mu- and delta-binding sites.4 In rat brain the capacity of the mu-binding site was 7.3 pmol/g brain, that of the delta-binding site 6.7 pmol/g brain and that of the kappa-binding site 2.0 pmol/g brain. Thus, the kappa-binding site had the lowest value whereas in the guinea-pig brain the capacity of the mu-binding site was lower than that of the delta- or kappa-binding site.

Animals↗
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↗
PubMed1981

Tobacco mosaic virus-enkephalin conjugates: potentiation of opioid activity.

Covalent tobacco mosaic virus-enkephalin analogue conjugates containing between 5 and 450 agonist molecules attached through their C-terminal amino acids were prepared and assayed in pharmacological and binding assays that distinguish between mu- and delta-receptors. Increases in peptide- and receptor-specific potency and affinity were observed with a certain preference of mu- over delta-receptors. The results may become important for mapping receptors or for isolating opiate receptor-bearing membrane vesicles.

Animals↗
PubMed1981

Some 14 beta-substituted analogues of N-(cyclopropylmethyl)normorphine.

A series of N-(cyclopropylmethyl)-14 beta-substituted-normorphine analogues was synthesized and tested for opioid agonist and antagonist activity in the guinea pig ileum and mouse vas deferens preparations. The 14 beta-bromo compound proved to be a pure antagonist equal in potency to naloxone in the guinea pig ileum assay. In contrast to N-(cyclopropylmethyl)-14 beta-hydroxynormorphine which was a pure antagonist, the corresponding sulfur analogue was about equal to nalorphine in agonist and antagonist potency.

Animals↗
PubMed1981

Characterization of the kappa-subtype of the opiate receptor in the guinea-pig brain.

1. In homogenates of guinea-pig brain, the characteristics of the binding of [3H]-ethylketazocine, an agonist for the putative kappa-receptor, were determined by estimation of the affinity and capacity of binding, by competitive inhibition for the binding site by unlabelled ligands and by selective protection of the binding site from alkylation by phenoxybenzamine. 2. At 25 degrees C the maximum number of binding sites for [3H]-ethylketazocine was about 14 pmol/g fresh brain, of which about 50% were high affinity sites. 3. In competition experiments, the high affinity binding of [3H]-ethylketazocine to the kappa-binding site was readily displaced by several kappa-agonists but not by the selective mu-ligand, D-Ala2, MePhe4, Gly-ol5-enkephalin or the selective delta ligand, D-Ala2, D-Leu5-enkephalin. In contrast, the kappa-agonists tested so far exhibit a high degree of cross-reactivity with the mu-binding site but somewhat less with the sigma-binding site. Similar specificities were observed in protection experiments. 4. The approximate proportions of the three subtypes of opiate receptor in the guinea-pig brain are 25% mu-binding sites, 45% delta-binding sites and 30% kappa-binding sites. 5. The endogenous opioids, Met-enkephalin, Leu-enkephalin and porcine beta-endorphin have only a low affinity for the kappa-binding site.

Animals↗