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Biomedical subjects

L E Robson

Publications and source records attributed to L E Robson.

33 records · Page 2Linked to original sources
PubMed1985

Kappa-binding and degradation of [3H]dynorphin A (1-8) and [3H]dynorphin A (1-9) in suspensions of guinea pig brain membranes.

Following incubation of [3H]dynorphin A (1-8) and [3H]dynorphin A (1-9) with suspensions of guinea pig brain membranes, analysis of the supernatants by HPLC has shown that both peptides are degraded at 25 degrees C and at 0 degrees C. Bestatin and captopril reduce degradation at 0 degrees C but for a similar degree of protection at 25 degrees C arginine-containing dipeptides are also required. The effects of these peptidase inhibitors on the degradation profiles indicate that [3H]dynorphin A (1-8) has three main sites of cleavage: the Tyr1-Gly2, Arg6-Arg7, and Leu5-Arg6 bonds. With [3H]dynorphin A (1-9) as substrate the Arg7-Ile8 and Ile8-Arg9 bonds are also liable to cleavage. In binding assays, in contrast to the effects of peptidase inhibitors on the degradation of unbound [3H]dynorphin A (1-8) and [3H]dynorphin A (1-9), bestatin and captopril have little effect on the binding characteristics of the tritiated dynorphin A fragments at the kappa-site at 0 degrees C. However, at 25 degrees C binding is low in the absence of peptidase inhibitors. When binding at mu- and delta-sites is prevented, the maximal binding capacities of [3H]dynorphin A (1-8), [3H]dynorphin A (1-9), and [3H](-)-bremazocine at the kappa-site are similar; [3H]dynorphin A (1-9) has 5-10 times higher affinity for the kappa-site than [3H]dynorphin A (1-8). Comparison of the effects of peptidase inhibitors on unbound dynorphin A fragments with their effects in binding assays suggests that the bound peptides are protected from the action of peptidases.

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

Opioid binding sites of the kappa-type in guinea-pig cerebellum.

(-)-[3H]Bremazocine interacts almost equally well with the mu-, delta- and kappa-types of opioid binding sites. In homogenates of guinea-pig cerebellum, it was bound with high affinity (KD = 0.046 nM) and the maximum binding capacity was 7.04 pmol/g wet wt of tissue. When the mu- and delta-binding of (-)-[3H]bremazocine was prevented with unlabelled ligands, a KD of 0.034 nM and a capacity of 5.94 pmol/g tissue was found for the kappa-binding site, which therefore comprised 84% of the opioid binding sites in the cerebellum. Autoradiographic analysis showed that the binding of (-)-[3H]bremazocine was relatively low in lobules IX and X and that it was predominantly located in the molecular and to a lesser extent in the granular layers. The addition of unlabelled mu- and delta-ligands did not alter the distribution. Thus, the guinea-pig cerebellum contains opioid binding sites of which almost all are of the kappa-type and is therefore an ideal tissue for the isolation of kappa-receptors and for the investigation of their biochemical and pharmacological properties.

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

[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↗
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↗
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↗
PubMed1979

Specific protection of the binding sites of D-Ala2-D-Leu5-enkephalin (delta-receptors) and dihydromorphine (mu-receptors).

Phenoxybenzamine causes a long-lasting inactivation of the opiate receptors of the mu- and delta-type in homogenates of guinea-pig brain. The effect is selectively prevented when, before exposure to phenoxybenzamine, the homogenate is pre-incubated with ligands of high affinity for either of the two binding sites, i.e. dihydromorphine for the mu-receptor and Tyr-D-Ala-Gly-Phe-D-Leu for the delta-receptor. In contrast, Tyr-D-Ala-Gly-Phe-L-Leu amide, which has high affinities for both binding sites, protects both receptor sites.

Animals↗
PubMed1979

The inhibitory effects of presynaptic alpha-adrenoceptor agonists on contractions of guinea-pig ileum and mouse vas deferens in the morphine-dependent and withdrawn states produced in vitro.

1 Isolated ilea from guinea-pigs implanted with morphine pellets were stimulated coaxially, either with or without morphine present in the bath fluid, and the longitudinal contractions recorded. 2 In the absence of morphine the inhibitory effects of the presynaptic alpha-adrenoceptor agonists, clonidine and oxymetazoline were much reduced and the dose-response curve was flat. This state of 'withdrawal' was readily reversed by morphine and levorphanol but not its inactive (+)-isomer, dextrophan. 3 The kappa-agonists, ketazocine and ethylketazocine, also restored the effects of clonidine as did the opioid peptides Tyr-D-Ala-Gly-Phe-D-Leu, acting preferentially on delta-receptors, and Tyr-D-Ala-Gly-MePhe-Met(O)-ol, acting mainly on micro-receptors. 4 The inhibitory effects of adrenaline and adenosine 3',5'-diphosphate were reduced at low but not at high concentrations. 5 In contrast, the inhibitory effect of clonidine on the electrically evoked contractions of vasa deferentia from mice implanted with morphine pellets was not abolished by the lack of morphine in the bath fluid or by addition of naloxone. 6 A possible explanation is suggested for the loss of the inhibitory effects of presynaptic alpha-adrenoceptor agonists in the withdrawn state of the dependent ileum.

Adenosine Diphosphate↗
PubMedDate not supplied

Safety and effect of transforming growth factor-beta(2) for treatment of venous stasis ulcers.

Transforming growth factor-beta(2) promotes healing in a variety of animal models and exhibits clinical effects thought to be mediated by connective tissue formation. Two clinical trials were conducted to evaluate the safety and effect of transforming growth factor-beta(2) purified from bovine bone and delivered topically to venous stasis ulcers three times per week for up to 6 weeks by means of a lyophilized collagen vehicle. The first was an open-label trial comparing transforming growth factor-beta(2) purified from bovine bone (0.5 microg/cm(2)) with a placebo consisting of lyophilized collagen vehicle-without active drug. After no safety issues arose in that trial, a prospectively randomized, closed-label, observer-blinded, three-armed trial was conducted to compare bovine transforming growth factor-beta(2) (2.5 microg/cm(2)) with the collagen matrix placebo vehicle and with a standard dressing. Standardized elastic compression was applied to all test extremities. The rate of reduction of ulcer area as measured by planimetry was the primary measure of effect. No serious safety-related events occurred in either trial. Clinical evaluation suggested that improvement in the quality and quantity of granulation tissue appeared to precede epithelialization of ulcers treated with bovine transforming growth factor-beta(2). In both studies, treatment with bovine transforming growth factor-beta(2) appeared to have a positive effect on the rate of ulcer closure, whereas ulcers in the control groups continued to exhibit impaired healing. In the open-label study, the mean rate of closure of ulcers treated with bovine transforming growth factor-beta(2) was significantly greater than that of ulcers treated with placebo. There was likewise enhanced reduction in ulcer area in the ulcers treated with bovine transforming growth factor-beta(2) in the second trial. However, because of a higher variability in patient response and a greater placebo effect, the difference was not significant. The placebo was not worse than the standard care arm, thereby showing that the vehicle is not injurious to healing. The combined results of the two trials suggest that, at doses of 0.5 to 2.5 microg/cm(2), bovine transforming growth factor-beta(2) is safe as a topically applied agent in a collagen matrix vehicle and can have a positive effect on closure of venous stasis ulcers. Large multicenter trials appear to be indicated to evaluate fully the potential utility of transforming growth factor-beta(2) in accelerating closure of chronic dermal ulcers.

Journal Article↗