Owen H. Callaghan

Studies of the in vitro human plasma degradation of methionine-enkephalin.

1. Incubation of [3H]tyrosine methionine-enkephalin (6 x 10(-9) M final concentration) with human platelet-poor plasma (1:9 ratio to Trizma Base buffer, pH 7.4) results mostly (greater than 95%) in hydrolysis of the tyrosyl-glycine peptide bond. This enzymatic reaction is essentially completed within 90 min, showing a half-life, Km and Vmax of 12.8 +/- 2.5 min, 0.70 +/- 0.01 mM and 17.90 +/- 1.05 mumol/L/min, respectively. These values are comparable to those previously reported for the human plasma degradation of leucine-enkephalin. 2. As expected hydrolysis of the methionine-enkephalin tyrosyl-glycine peptide bond was blocked by the known aminopeptidase inhibitors bestatin and puromycin (IC50 1.2 +/- 0.4 and 4.3 +/- 2.4 microM, respectively) but not by either thiorphan or captopril. 3. Neither the storing (up to 60 days) nor the freezing and thawing (up to ten times during a 60 days periods) significantly changed the above kinetic parameters, showing the stability of the plasma methionine-enkephalin degrading aminopeptidase.

Platelet monoamine oxidase activity and plasma levels of non-catecholic phenylethylamines in insulin-dependent diabetic subjects

The activity of blood platelets monoamine oxidase (MAO) was significantly reduced in a group of insulin-dependent diabetics when compared to sex- and age-matched controls. This enzymatic change was accompanied by a dramatic increase in the plasma levels of phenylethylamine, whereas no significant changes were observed for the concentration of either p-tyramine or phenylethanolamine. Levels of the o- and m-isomers of tyramine were below detectable limits (less than 0.050 ng/ml). A possible role of the MAO/monoamine system in the pathophysiology of diabetes is discussed.

The Kinetics of Hydroxylation of Phenylethylamine, Amphetamine and Phenylalanine in Rodent Tissues

Phenylethylamine (PEA) and amphetamine (α-MePEA) are closely related structurally and pharmacologically. Both amines elicit a number of similar clinical and behavioral (animal) responses, appear to use similar transport systems and to act via some common neurochemical mechanisms (1, 2). These observations have led several investigators to suggest that at least some of the α-MePEA actions are mediated via PEA, an endogenous amine (3) which has been conceptualized as a naturally occurring α-MePEA (4). We now report for the first time that both amines are p-hydroxylated, in vitro, by a common rat liver microsomal enzyme. It seems likely that PEA and α-MePEA might compete for the same catalytic site.

Changes in plasma methionine-enkephalin levels associated with a cluster headache episode.

Eighteen male cluster headache (CH) inpatients within a CH series participated in this research. Blood samples were drawn from patients at least 6-hour pain-free after the last acute CH episode and then shortly prior (SP), during, and soon after (SA) a new acute CH attack. Three healthy male, age-comparable drug-free volunteers served as controls; 5 samples were obtained from each of these individual over a 24-hour period. Individual patient’s methionine-enkephalin (MET) plasma concentration showed significant changes, and in some subjects, dramatic changes, during the different phases of a single CH episode. Peptide levels followed a general pattern of higher plasma concentration SP to an acute CH attack, followed by decreased levels during the attack itself, and falling even further SA the acute episode. Consistently, 16 of the 18 patients tested showed pre-CH peptide levels significantly higher (arbitrarily the authors considered values 20% or more as “significant”) than their own values obtained during the acute CH pain phase, with observed differences reaching 80% or more in 7 of these individuals. For about half of these patients, peptide concentration during the acute CH episode was significantly above the control’s range (68.2–87.6 pg MET/mL; controls circulating MET concentration remaining essentially unchanged during a 24-hour period). MET levels were further decreased in essentially all of the post-CH samples, with values falling within (n = 6) or even further below than those in the control’s range (n = 11). Neither age, time of CH occurrence, nor patient’s use of a number of medications known for failing to influence plasma MET degradation kinetics seemed to significantly influence MET levels. These results might help in the biochemical characterization of the actual phases of a CH episode. Developing drugs modulating MET bioavailability could lead to novel antinociceptive agents useful for the treatment of CH’s associated pain.

Hydroxylation of phenylethylamine by rat liver preparations. Inhibition studies.

1. Rat liver 100,000 g pellet microsomal fraction p-hydroxylate phenylethylamine to tyramine in a relatively slow proceeding, NADPH-requiring reaction; Km 2.1 x 10(-5) M and Vmax 0.32 nmol/mg protein/20 min. 2. This reaction is inhibited, either competitively, noncompetitively or uncompetitively by a number of behaviorally active monomethylated and monohalogenated derivatives of phenylethylamine. 3. Whereas formation of tyramine was not significantly affected by L-phenylalanine or its p-chloro derivative, it was competitively inhibited by imipramine, iprindole and alprazolam. 4. It is suggested that at least some of the effects of these drugs may result from their ability to interfere with phenylethylamine metabolism.

Properties of rabbit antiragweed and antihorseradish peroxidase homocytotropic antibodies

Abstract Homocytotropic antibodies (HCA) were produced by the rabbit after primary injection of the dialysis residue of an aqueous extract of dwarf ragweed pollen (DRg) or a purified dwarf ragweed pollen extract (Pool C) only when the antigen was adsorbed on Al(OH) 3 . The properties of rabbit ragweed-specific HCA were qualitatively similar to ragweed skin-sensitizing antibodies found in sera of ragweed-allergic human beings, namely: homocytotropic activity (72 hour homologous PC A reaction), heat lability at 56 °C., and inactivation by reduction with 2-mercaptoethanol followed by alkylation with iodoacetamide. The molecular weight of rabbit HCA was ~250,000 (Sephadex G-200), somewhat higher than human γE skin-sensitizing antibodies. Most rabbits immunized with ragweed antigen-Al(OH) 3 systems gave positive conjunctival and intradermal reactions. Intravenous challenge (8 mg. per kilogram) of these animals produced anaphylaxis. HCA were also elicited in response to the immunologic marker, horseradish peroxidase (HRP), when admixed with Al(OH) 3 . The properties of HRP-specific HCA (molecular weight ~250,000) were similar to rabbit ragweed-specific HCA. The results suggest that the rabbit can be used as a model to study the biosynthesis of antibodies that have some physicochemical and biologic characteristics in common with human antibodies involved in immediate hypersensitivity.

Phenylethylamine metabolism to tyramine by postmortem human brain preparations

Human brain preparations obtained from either the putamen, thalamus, hippocampus or lateral occipital gyrus p-hydroxylate phenylethylamine to tyramine, a reaction carried out by a microsomal (100,000 xg pellet) membrane bound, NADPH-requiring enzyme. This is a minor metabolic pathway occurring in chronic psychiatric patients, as well as in age-comparable controls.

Uptake of Carbohydrate by Polyribosomes During the Immune Response to Ragweed Antigen

The in vitro radioactive labeling of polyribosomes in slices of rabbit lymph glands, liver and other tissues engaged in glycoprotein synthesis was studied by sucrose density gradien