Victor Brantl

Psychotomimesis mediated by kappa opiate receptors

The kappa opioid agonists are analgesics that seem to be free of undesired morphine-like effects. Their dysphoric actions observed with the kappa agonist cyclazocine are thought to be mediated by an action at sigma-phencyclidine receptors. The benzomorphan kappa agonist MR 2033 is inactive at sigma-phencyclidine receptors. In male subjects, the opiate-active (-)-isomer, but not the (+)-isomer, elicited dose-dependent dysphoric and psychotomimetic effects that were antagonized by naloxone. Thus, kappa opiate receptors seem to mediate psychotomimetic effects. In view of the euphorigenic properties of mu agonists, our results imply the existence of opposed opioid systems affecting emotional and perceptual experiences.

Opioid activities of β-casomorphins

Abstract β-Casomorphin-7 (H-Tyr-Pro-Phe-Pro-Gly-Pro-Ile-OH) and its analogues: β-casomorphin-6, (-5) and (-4) (derived by sequential removal of respectively one, two or three amino acid residues from the C-terminus), were tested for their opioid activities in a variety of assay systems. Each of the four peptides displayed opioid activity in an opiate receptor binding assay, the isolated mouse vas deferens (MVD), the guinea-pig ileum longitudinal muscle myenteric plexus preparation (GPI) and produced naloxone-reversible analgesia after intracerebroventricular injection into rats. In contrast, none of the peptides displayed opioid activity in the isolated rat vas deferens preparation (RVD). β-Casomorphin-5 was the most potent compound in all the assays employed. Each β-casomorphin was more potent on the GPI than on the MVD. In view of the fact that the GPI, MVD and RVD are populated predominantly by μ-, δ- and e-receptors, respectively, the β-casomorphins probably represent μ-type opiate receptor agonists.

Antinociceptive potencies of β-casomorphin analogs as compared to their affinities towards μ and δ opiate receptor sites in brain and periphery

Abstract β-Casomorphins and their analogs were tested for their opioid activities in the myenteric plexus longitudinal muscle preparation of the guinea pig ileum (GPI), the isolated mouse vas deferens (MVD), and for their affinities to μ- δ- and κ-binding sites in rat brain membranes. C-terminal amidation of β-casomorphin-4 and (-5) increased opioid potency in both organ preparations (GPI, MVD) and affinity to μ-binding sites in brain whereas binding to δ-sites was diminished. These β-casomorphin-amides displayed a 2–3 times greater naloxone reversible antinociceptive effect than natural β-casomorphins. Introduction of D-alanine at position 2 in the β-casomorphin-amides increased potency in the GPI whereas activity in the MVD was only slightly changed. These compounds, however, showed a remarkable increase in binding to δ-sites in brain with an unaffected or slightly increased binding to μ-sites and decreased binding to κ-sites. D-Ala2-β-casomorphin-4 and (-5) amides were 10 times more potent antinociceptive agents than corresponding β-casomorphin-amides. These results suggest firstly, that peripheral δ-receptors in the MVD are not as closely related to δ-binding sites at rat brain membranes as is the case with μ-receptors in the GPI and μ-binding sites, and secondly, in addition to μ-receptors, δ-receptors may be of importance in mediating antinociception.

Opioid receptor affinities of the blood-derived tetrapeptides hemorphin and cytochrophin

Hemorphin-4 and cytochrophin-4 displayed affinities for mu- and delta-opioid receptors that were in the same range as those observed for the structurally related beta-casomorphins. However, they showed markedly higher affinities at kappa-opioid binding sites when compared to the beta-casomorphins. These blood-derived peptides could be involved in blood pressure regulation.

Hemorphins, cytochrophins, and human beta-casomorphins bind to antiopiate (Tyr-MIF-1) as well as opiate binding sites in rat brain

Novel peptides with opiate activity, derived from endogenous sources (human and bovine casomorphins from milk, hemorphins from hemoglobin, and cytochrophins from mitochondrial cytochrome b), were tested for their ability to inhibit binding of the brain peptide Tyr-MIF-1 (Tyr-Pro-Leu-Gly-NH2) to its high affinity sites in rat brain. The order of potency in inhibiting binding of 125I-Tyr-MIF-1 was: hemorphin and bovine casomorphins greater than Tyr-MIF-1 greater than cytochrophins greater than human casomorphins. Naloxone and DAMGO were ineffective at inhibiting Tyr-MIF-1 binding. The results provide evidence that, in addition to their ability to bind to mu opiate receptors, these novel endogenous peptides with opiate activity and a peptide (Tyr-MIF-1) with antiopiate properties also bind to a non-opiate site labeled by Tyr-MIF-1. These sites could be involved in a balance between opiate and antiopiate peptides.

Effect of opiate-active substances on pancreatic polypeptide levels in dogs

The present study examines the effect of orally and intravenously administered opiate-active substances on peripheral vein plasma pancreatic polypeptide (PP) levels in conscious dogs. The intragastric instillation of digested gluten stimulated postprandial PP levels significantly which was reduced by the specific opiate-receptor antagonist naloxone. Naloxone had no effect when added to undigested gluten. Similarly, naloxone reduced significantly the postprandial PP response to a test meal of casopeptone which contains the opiate-active β-casomorphins. The addition of synthetic β-casomorphins to a liver extract/sucrose test meal significantly augmented the rise of postprandial PP levels which was also blocked by naloxone. The intravenous infusion of morphine, leu-enkephalin, D-ala2-D-leu5-enkephalin, β-casomorphin-5 and β-casomorphin-4 elicited a dose-dependent and naloxone reversible effect on basal PP levels. During a background infusion of glucose and amino acids the same opiate-active substances had either none or a stimulatory effect on PP release in these dogs. The addition of naloxone abolished the stimulatory effect in response to β-casomorphin-5 and β-casomorphin-4 and resulted in an inhibition of PP levels during the infusion of morphine and leu-enkephalin. This latter inhibitory effect was no longer observed when the dose of naloxone was increased ten- and fifty-fold, respectively. The present data suggest that orally ingested opiate-active substances participate in the stimulation of postprandial PP release in dogs via specific opiate-receptor mediated mechanisms. The effect of intravenously administered opiate-active substances on PP levels depends on the metabolic state with regard to the level of circulating nutrients. It is suggested that PP release is stimulated via μ-opiate receptors and inhibited via δ-opiate receptors. An increase of circulating nutrients would “activate” μ-receptor sites which are masked in the basal state when exogenous opiates are administered. However, with regard to endogenous opiates an increase of circulating nutrients, mainly carbohydrates, activates inhibitory effects of endogenous opiates suggesting that exogenous and endogenous opiates act at different target sites.

β-CASOMORPHINS–NOVEL OPIOID PEPTIDES DERIVED FROM BOVINE CASEIN–ISOLATION AND STRUCTURE

Material, showing opioid activity in the guinea pig ileum bioassay, could be obtained by chloroform-metha-nol extraction of casein peptone. The extract was purified by adsorption procedures, high pressure liquid chromatography and gel filtration. Several opioid components could be separated. One of these accounted for over 90% of the total opioid activity and was subjected to structure analysis. It contained a pure heptapeptide, Tyr-Pro-Phe-Pro-Gly-Pro-11e. Evidence for its identity with the opioid principle was obtained by measuring changes in activity on acid hydrolysis, acetylation and enzymatic digestion, all of which altered the peptide structure; in all cases the activity also disappeared. The peptide is highly resistant towards most proteolytic enzymes, even pronase. However, carboxypeptidase γ degrades it, first giving rise to a pentapeptide, Tyr-Pro-Phe-Pro-Gly, which is more active than the heptapeptide, and then a tripeptide, Tyr-Pro-Phe, which is inactive. The sequence of the heptapeptide identified it as a fragment of bovine β-casein. Because of this the opioid peptide was called β-casomorphin.

Effects of oral casokefamide on plasma levels, tolerance, and intestinal transit in man.

Food-derived opioid peptides such as beta-casomorphins are of interest for treatment of chronic diarrhea. The beta-casomorphin analog casokefamide was administered orally at doses of 5.5, 8.0, and 16.0 mg to 10 healthy male volunteers, respectively. Dose-dependent increases of plasma levels with a maximum of 350 fmol/l were determined. No side-effects due to casokefamide has been observed. In comparison to placebo, casokefamide showed a trend toward prolongation of oro-caecal transit time. Orally applied casokefamide is well tolerated and may represent a useful tool for treatment of diarrhea in the future.

Role of endogenous opiates in postprandial somatostatin release

Previously, we have demonstrated the effects of exogenously administered opiates on somatostatin release in dogs and therefore the present study was designed to determine the effect of endogenous opiates via naloxone-induced opiate receptor blockade on somatostatin release. Additionally, plasma insulin and pancreatic polypeptide (PP) levels were determined in response to intragastrically instilled protein, carbohydrate and fat test meals in a group of eight conscious dogs. To all test meals either naloxone (4 mg) or saline was added. The rise of plasma somatostatin levels in response to liver extract, sucrose and fat was attenuated significantly by naloxone. Naloxone had no effect on the rise of postprandial plasma insulin and PP levels. The present data demonstrate that endogenous opiates have a stimulatory effect on postprandial somatostatin release in dogs which indicates a tight interaction that might be of relevance for nutrient homeostasis.