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Biomedical subjects

H W Kosterlitz

Publications and source records attributed to H W Kosterlitz.

At least 19 recordsLinked to original sources
PubMed1991

Opioid activity of dermenkephalin analogues in the guinea-pig myenteric plexus and the hamster vas deferens.

1. To elucidate the structural features required for selective and potent action of dermenkephalin at the delta-opioid receptor, a series of analogues of dermenkephalin and dermorphin were tested for their effectiveness in depressing electrically-evoked contractions of the vas deferens of the hamster (delta-opioid receptors) and the guinea-pig myenteric plexus-longitudinal muscle preparation (mu- and kappa-opioid receptors). 2. Dermenkephalin was more selective and more potent at delta-receptors than the delta-ligand [D-Pen2, D-Pen5]-enkephalin. The responses to dermenkephalin in the hamster vas deferens were increased by addition of peptidase inhibitors; the maximum effect was obtained with 3 microM thiorphan. 3. [L-Met2]-dermenkephalin had 0.2% and [L-Ala2]-dermorphin 0.01% of the agonist activity of the corresponding endogenous peptides which have D-amino acids in position 2. The pharmacological activity of these analogues was unaffected by inhibition of peptidases. This emphasizes the role that the D-configuration plays in determining the bioactive folding of these highly active peptides. 4. Dermenkephalin-(1-6)-NH2 was more potent at delta-receptors than at mu-receptors whereas, dermenkephalin-(1-4)-NH2 is a selective mu-agonist, having no activity at delta-receptors. 5. Substitution of the C-terminal tripeptide of dermorphin with the C-terminal tripeptide of dermenkephalin abolished the mu-receptor preference of dermorphin. The resulting hybrid peptide, Tyr-D-Ala-Phe-Gly-Leu-Met-Asp-NH2 was as potent as dermenkephalin at delta-receptors. A shift towards a preference for delta-receptors was obtained when the C-terminal tetrapeptide of dermorphin was replaced by the C-terminal tetrapeptide of dermenkephalin. 6. Substitution of Asp by Asn in position 7 of dermenkephalin caused an increase in mu-receptor potency and a decrease in delta-receptor potency, resulting in a 20 fold decrease in mu-receptor selectivity. Dermenkephalin-(1-6)-NH2 and [Asn7]-dermenkephalin have almost identical delta-receptor agonist potencies and ratios of IC50 in the myenteric plexus to IC50 in the hamster vas deferens. 7. The results obtained emphasise the importance of a negative charge at the C-terminus of dermenkephalin for selectivity at the delta-opioid receptor. Furthermore, the hydrophobic residues Leu5 and Met6 may be critical in ensuring tight binding to the receptor which results in high agonist potency.

Amino Acid Sequence↗
PubMed1991

Electrically-induced release of opioid peptides from the guinea-pig myenteric plexus preparation.

Preparations of guinea-pig myenteric plexus-longitudinal muscle suspended in Krebs solution were stimulated electrically in the presence of cycloheximide and tetraethylammonium. The amounts of eleven endogenous opioid peptides released into the perifusing Krebs solution were determined and correlated with the decrease in the tissue contents induced by stimulation. For pro-enkephalin fragments the ratio of release to reduction in tissue contents was 29 to 43% for [Met]enkephalin, [Leu]enkephalin, [Met]enkephalyl-RF and [Met]enkephalyl-RGL. With [Met]enkephalyl-RRV-NH2 (BAM-8) the ratio was higher by 50% or more. However, it is of interest that there was no release of the probable precursor [Met]enkephalyl-RRVGRPEWWMDYQ(BAM-18). In this context it may be important that BAM-8 is the only endogenous opioid peptide having -NH2 at the C-terminal. The low tissue levels of pro-dynorphin derived peptide have made estimation of release unreliable.

Amino Acid Sequence↗
PubMed1990

Effect of Tris, HEPES, and TES buffers on binding at mu-, delta-, and kappa-opioid sites in guinea pig brain.

In homogenates of guinea-pig brain minus cerebellum, the delta-binding of 1.5 nM [3H]-[D-Pen2,D-Pen5]enkephalin is little affected by the type and concentration of the three buffers, Tris-HCl, HEPES-KOH, or TES-KOH (10-75 mM). However, the mu-binding of 1 nM [3H]-[D]Ala2,MePhe4,Gly-ol5]enkephalin or the kappa-binding of 1.5 nM [3H]-U-69,593 is influenced by the choice of buffer. A suitable concentration of buffer for further analyses of opioid binding has been found to be 10 mM. At each site, the effects of MgCl2 on binding are the same whether 10 mM Tris-HCl, HEPES-KOH, or TES-KOH are used but variations of the effects of NaCl confirm the view that mu- and kappa-sites, but not delta-sites, are affected by choice of buffer. Furthermore, under some assay conditions the effects of NaCl and MgCl2 at the kappa-sites of guinea-pig cerebellum differ from their effects in brain minus cerebellum, indicating that these are differences of binding characteristics at the kappa-sites of these tissues.

Animals↗
PubMed1989

Regional variations in binding capacities at mu-, delta- and kappa-opioid sites in membrane suspensions from rabbit brain.

The highest maximum binding capacities at the mu-sites of rabbit brain are in the striatum, with intermediate levels in the diencephalon, mesencephalon, cerebellum and cortex and low levels in the pons-medulla and hippocampus. For the delta-site the highest maximum binding capacity is also in the striatum; there are almost equally low levels in the other brain regions. At the kappa-sites the maximum binding capacities are highest in the diencephalon; there are intermediate levels in the cortex and striatum, and low levels in the mesencephalon, cerebellum, hippocampus and pons-medulla. The KD values lack reproducibility; there are no regional variations at the kappa-site as estimated with [3H](-)-bremazocine, but the possibility cannot be excluded that there are regional variations in the KD values for [3H][D-Ala2,MePhe4,Gly-ol5]enkephalin at the mu-site or for [3H][D-Ala2,D-Leu5]enkephalin at the delta-site. It may be important to use saturation analysis in future investigations of the distributions of the binding sites.

Analgesics↗
PubMed1988

Cation dependence of opioid receptor binding supports theory on membrane-mediated receptor selectivity.

A quantitative analysis of the binding of dynorphin A-(1-9)-nonapeptide to the opioid Kappa-receptors of the guinea pig cerebellum [Paterson et al. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 6216-6220] shows that changes in electrostatic surface accumulation of the ligand fully account for the observed suppression of binding by a series of univalent and divalent salts. Binding to mu- and delta-receptors, on the other hand, is subject to additional ion-specific effects. These observations support the membrane locations for the receptor sites proposed by the "membrane compartments" theory for opioid receptor selection.

Algorithms↗
PubMed1988

Tissue content of opioid peptides in the myenteric plexus-longitudinal muscle of guinea-pig small intestine.

We have developed a method that is based on two HPLC systems and permits the separation of endogenous opioid peptides in tissue extracts. The individual peptides are bioassayed on the mouse isolated vas deferens; naloxone (100 nM) ensures opioid specificity. In the myenteric plexus-longitudinal muscle preparation of the guinea-pig small intestine, the tissue content of prodynorphin-derived peptides is lower than those of proenkephalin-derived peptides. No beta-endorphin was detected. Of the prodynorphin fragments, alpha-neoendorphin, beta-neoendorphin, dynorphin A(1-8), and dynorphin B are present in equimolar concentrations (12-15 pmol/g) whereas the tissue content of dynorphin A is only 0.8 pmol/g. Processing of proenkephalin leads to at least six opioid peptides. The tissue contents of [Leu5]enkephalin, [Met5]enkephalyl-Arg-Gly-Leu, and [Met5]enkephalyl-Arg-Phe are 90-100 pmol/g and the content of [Met5]enkephalin is 405 pmol/g. BAM-18 and [Met5]enkephalyl-Arg-Arg-Val-NH2 are present in much lower concentrations, 24 and 5 pmol/g, respectively. Although present in low amounts, BAM-18 and [Met5]-enkephalyl-Arg-Arg-Val-NH2 have high affinity for the mu-opioid binding site and to a lesser extent for the kappa-site; this binding profile differs from that of the other proenkephalin fragments all of which have high affinities for the mu- and delta-sites.

Animals↗
PubMed1988

Modulation of binding at opioid receptors by mono- and divalent cations and by cholinergic compounds.

In membrane suspensions from guinea-pig brain, NaCl, LiCl, NH4Cl and KCl, inhibit the equilibrium binding (25 degrees C) of the selective mu-agonist [3H]-[D-Ala2,MePhe4,Gly-ol5]enkephalin, the selective delta-agonist [3H]-[D-Pen2,D-Pen5]enkephalin and the selective kappa-agonist [3H]-dynorphin A (1-9). Choline chloride inhibits the binding of the mu- and kappa-agonists but not of the delta-agonist; the choline derivative, methacholine, inhibits also the binding of the delta-agonist. Binding of the delta-agonist is potentiated by CaCl2, MgCl2 and MnCl2; these salts inhibit binding of the kappa-agonist. As far as binding of the mu-agonist is concerned, MgCl2 and MnCl2 may potentiate or inhibit whereas CaCl2 is only inhibitory. The binding of the mu-antagonist [3H]-naloxone is potentiated by NaCl; while the threshold of inhibition by LiCl is increased there is no potentiation. In membrane suspensions of the rabbit cerebellum about 80% of the opioid binding sites are of the mu-type; the binding of the mu-agonist [3H]-[D-Ala2,MePhe4,Gly-ol5]enkephalin is inhibited by NaCl, LiCl, KCl and choline chloride whereas that of the mu-antagonists [3H]-naloxone and [3H]-(-)-bremazocine is potentiated at low concentrations but inhibited at higher concentrations of NaCl. In membranes of the guinea-pig cerebellum about 80% of the opioid binding sites are of the kappa-type; they are particularly effective for assays of kappa-receptors when the selective kappa-agonist [3H]-dynorphin A (1-9) is used as ligand.

Animals↗
PubMed1987

Diprenorphine has agonist activity at opioid kappa-receptors in the myenteric plexus of the guinea-pig ileum.

The opioid agonist and antagonist activities of diprenorphine have been tested in four in vitro bioassay preparations. Diprenorphine is an antagonist at delta-receptors in the hamster vas deferens, at mu-receptors in the rat vas deferens and at kappa-receptors in the rabbit vas deferens. In the guinea-pig ileum it is an antagonist at mu-receptors and an agonist at kappa-receptors.

Animals↗
PubMed1987

Differential postnatal development of mu-, delta- and kappa-opioid binding sites in rat brain.

With selective labelling techniques and analysis of saturation curves it is shown that in rat brain the concentrations of mu-, delta- and kappa-binding sites increase differentially during postnatal development. There are no changes in the binding affinities. The concentration (pmol/g brain) of kappa-sites are first to reach adult levels, namely between 7 and 14 days after birth. Adult levels of mu-sites are attained between 14 and 21 days after birth. The most striking finding is that development of delta-sites, which are not detectable 3 days after birth by the method used, lags markedly behind that of mu- and kappa-sites. The profile of development in rat brain is compared to that found previously in mouse brain.

Animals↗
PubMed1987

Effects of cations on binding, in membrane suspensions, of various opioids at mu-sites of rabbit cerebellum and kappa-sites of guinea-pig cerebellum.

At the mu-sites of rabbit cerebellum, NaCl, LiCl, KCl, choline chloride and MnCl2 were tested for potentiation and inhibition of the binding of several opioids. Naloxone, (-)-bremazocine and diprenorphine are mu-antagonists in pharmacological assays and their binding is potentiated by the lower concentrations and inhibited by the higher concentrations of NaCl. The binding of the agonists [3H]-[D-Ala2, MePhe4, Gly-ol5]enkephalin and [3H]-dihydromorphine is inhibited. MnCl2 potentiates the binding of the agonist [3H]-[D-Ala2, MePhe4, Gly-ol5]enkephalin but not the binding of the antagonists. The thresholds of inhibition and slopes of the dose-response curves for inhibition by MnCl2 and LiCl vary. This finding may indicate that potentiating effects of MnCl2 and LiCl are masked by simultaneous inhibition. At the kappa-sites of guinea-pig cerebellum, NaCl, KCl and MnCl2 inhibit the binding of [3H]-dynorphin A (1-8), [3H]-dynorphin A (1-9), [3H]-(-)-bremazocine, [3H]-tifluadom, and [3H]-diprenorphine. NaCl also causes a small potentiation of the binding of [3H]-diprenorphine, which is a kappa-agonist in the guinea-pig myenteric plexus but a kappa-antagonist in the rabbit vas deferens. The slopes of the inhibitory dose-response curves and the thresholds of inhibition vary with the different ligands. Therefore some potentiating effects may have been masked. The results support the view that NaCl, and perhaps LiCl, but not KCl and choline chloride, potentiate the binding of mu-antagonists but not the binding of mu-agonists. It is not yet possible to decide whether, at the kappa-site, there is a similar differentiation of the binding of agonists and antagonists.

Animals↗
PubMed1986

Control by cations of opioid binding in guinea pig brain membranes.

In membrane suspensions from guinea pig brain or cerebellum, NaCl, LiCl, NH4Cl, and KCl inhibit the equilibrium binding at 25 degrees C of the selective mu-agonist [3H][2-D-alanine,4-methylphenylalanine,5-glycinol]enkephalin ([D-Ala2,MePhe4,Gly-ol5]EK), the selective delta-agonist [3H][2-D-penicillamine,5-D-penicillamine]enkephalin ([D-Pen2,D-Pen5]-EK), and the selective kappa-agonist [3H]dynorphin A-(1-9). Choline chloride inhibits mu- and kappa-binding but not delta-binding. The relative activities of these monovalent salts and the slopes of the dose-response curves are site-dependent. Binding at the kappa-binding site is also inhibited by CaCl2, MnCl2, and MgCl2. On the other hand, these divalent salts potentiate delta-binding, and MnCl2 and MgCl2 have both potentiating and inhibitory effects on mu-binding; CaCl2 inhibits but does not potentiate mu-binding. Thus, the mechanisms by which monovalent cations inhibit opioid binding differ from those of divalent cations, and the mechanisms of action of both monovalent and divalent cations may differ at each site. When the antagonist [3H]naloxone, rather than the agonist [3H][D-Ala2,MePhe4,Gly-ol5]EK, is used to label the mu-binding site, the main effect of NaCl is to potentiate binding; a 22-fold higher concentration of LiCl is required to inhibit binding. The effects of NH4Cl, KCl, MnCl2, MgCl2, CaCl2, and choline chloride are little changed when [3H]naloxone is the ligand.

Ammonium Chloride↗
PubMed1986

Bremazocine is an agonist at kappa-opioid receptors and an antagonist at mu-opioid receptors in the guinea-pig myenteric plexus.

The agonist and antagonist activity of bremazocine at opioid receptors in the guinea-pig myenteric plexus preparation was determined in untreated tissues and in tissues in which either mu-9 or kappa-opioid receptors were blocked preferentially. After pretreatment of the tissue with beta-funaltrexamine for 90 min followed by washing out, the IC50 value of the selective mu-ligand [D-Ala2,MePhe4,Gly-ol5]enkephalin was increased 67 fold whereas the IC50 values of the selective kappa-ligand U-69,593 and of the non-selective kappa-ligand bremazocine were not significantly changed. In this experimental design bremazocine acted only on kappa-receptors. After pretreatment of the tissue with beta-chlornaltrexamine and 10 microM of the mu-ligand for 30 min followed by washout, the IC50 value of the mu-ligand was increased 2 fold whereas the IC50 value of the selective kappa-ligand was increased 32 fold and that of bremazocine 62 fold. Under these experimental conditions, it was shown that bremazocine is an antagonist against [D-Ala2,MePhe4,Gly-ol5]enkephalin at the mu-receptor (Ke = 1.6 nM). The residual agonist activity of bremazocine is at the kappa-receptor. In naive myenteric plexus preparations the mu-antagonist activity of bremazocine cannot be demonstrated because its potency at the kappa-receptor is very high. This dual action may be of importance for the responses of bremazocine in other peripheral and central tissues.

Animals↗
PubMed1986

Site-dependent effects of ions on mu-, delta- and kappa-opioid binding in suspensions of guinea-pig brain membranes.

NaCl, LiCl, NH4Cl and KCl inhibit the equilibrium binding of peptide agonists at each of the mu-, delta- and kappa-sites; the orders of potencies and the slopes of the dose-response curves are site-dependent. In contrast, the monovalent salt choline chloride inhibits mu- and kappa-binding but has little effect on delta-binding. The profiles of activity of MnCl2, CaCl2 and MgCl2 are also site-dependent but differ markedly from those of the monovalent salts.

Ammonium Chloride↗