Christopher J. Fowler

The effect of age on the activity and molecular properties of human brain monoamine oxidase

The effect of age upon monoamine oxidase -A and -B (MAO-A and -B) in 23 different regions of human brain was determined. There was a significant positive correlation with age in 19 out of 23 regions for MAO-B, but no positive correlation with age was found for MAO-A. The increased MAO-B activity was found, in 5 out of 5 regions tested, to be due entirely to an increased enzyme concentration, rather than due to an increased molecular turnover number of the enzyme. The responses of the mitochondrial marker enzymes succinate dehydrogenase (SDH) and malate dehydrogenase (MDH) were studied in 5 brain regions, and no consistent change in activity found with age. The lysosomal enzyme acid phosphatase was found to tend towards an increased activity with age. No difference in either the specific activities or molecular characteristics of MAO were found between men and women. Cross-correlation studies of the data, after compensation for the effects of age, indicated that the activities of the two enzyme forms are under some form of organized control across the whole brain. Such a finding is consistent with a genetic regulation of the enzyme forms.

The palmitoylethanolamide family: a new class of anti-inflammatory agents?

The discovery of anandamide as an endogenous ligand for the cannabinoid receptors has led to a resurgence of interest in the fatty acid amides. However, N-palmitoylethanolamine (PEA), a shorter and fully saturated analogue of anandamide, has been known since the fifties. This endogenous compound is a member of the N-acylethanolamines, found in most mammalian tissues. PEA is accumulated during inflammation and has been demonstrated to have a number of anti-inflammatory effects, including beneficial effects in clinically relevant animal models of inflammatory pain. It is now engaged in phase II clinical development, and two studies regarding the treatment of chronic lumbosciatalgia and multiple sclerosis are in progress. However, its precise mechanism of action remains debated. In the present review, the biochemical and pharmacological properties of PEA are discussed, in particular with respect to its analgesic and anti-inflammatory properties.

Monoamine oxidase A and B: A useful concept?

The use of a number of inhibitors of monoamine oxidasc (monoamine:O, oxidoreductase, EC 1.4.3.4) has shown that many mam~lian tissues contain two major forms of the enzymic activity that differ in their substrate specificities and sensitivities to inhibitors. Although several pieces of evidence indicate that these two forms may not represent two distinct enzymes but result from a single enzyme species existing in different membrane-bound environments [see eg., 1.21. it has frequently been assumed that their specificities will be broadly similar in a variety of organs and tissues and also in different animal species. If such an assumption were true it should be possible to extrapolate results found in any convenient laboratory animal to the situation in man. It would seem however, that this general assumption may be an oversimplification. This review is therefore an attempt to assess the usefulness of the concept of the existence of two functionally distinct forms of the enzyme, without exploring the possible nature of any such forms, since this aspect has recently been reviewed in detail elsewhere f-?].

Inhibitors of fatty acid amide hydrolase reduce carrageenan-induced hind paw inflammation in pentobarbital-treated mice: comparison with indomethacin and possible involvement of cannabinoid receptors

The in vivo effect of inhibitors of fatty acid amide hydrolase (FAAH) upon oedema volume and FAAH activity was evaluated in the carrageenan induced hind paw inflammation model in the mouse. Oedema was measured at two time points, 2 and 4 h, after intraplantar injection of carrageenan to anaesthetised mice. Intraperitoneal (i.p.) injections of the FAAH inhibitor URB597 (0.1, 0.3, 1 and 3 mg kg−1) 30 min prior to carrageenan administration, dose‐dependently reduced oedema formation. At the 4 h time point, the ED50 for URB597 was ∼0.3 mg kg−1. Indomethacin (5 mg kg−1 i.p.) completely prevented the oedema response to carrageenan. The antioedema effects of indomethacin and URB597 were blocked by 3 mg kg−1 i.p. of the CB2 receptor antagonist SR144528. The effect of URB597 was not affected by pretreatment with the peroxisome proliferator‐activated receptor γ antagonist bisphenol A diglycidyl ether (30 mg kg−1 i.p.) or the TRPV1 antagonist capsazepine (10 mg kg−1 i.p.), when oedema was assessed 4 h after carrageenan administration. The CB1 receptor antagonists AM251 (3 mg kg−1 i.p.) and rimonabant (0.5 mg kg−1 i.p.) gave inconsistent effects upon the antioedema effect of URB597. FAAH measurements were conducted ex vivo in the paws, spinal cords and brains of the mice. The activities of FAAH in the paws and spinal cords of the inflamed vehicle‐treated mice were significantly lower than the corresponding activities in the noninflamed mice. PMSF treatment almost completely inhibited the FAAH activity in all three tissues, as did the highest dose of URB597 (3 mg kg−1) in spinal cord samples, whereas no obvious changes were seen ex vivo for the other treatments. In conclusion, the results show that in mice, treatment with indomethacin and URB597 produce SR144528‐sensitive anti‐inflammatory effects in the carrageenan model of acute inflammation.

'Entourage' effects of N-acyl ethanolamines at human vanilloid receptors. Comparison of effects upon anandamide-induced vanilloid receptor activation and upon anandamide metabolism

The abilities of a series of saturated N‐acyl ethanolamines and related compounds to affect the ability of anandamide (AEA) to produce a Ca2+ influx into human embryonic kidney cells expressing the human vanilloid receptor (hVR1‐HEK293 cells) has been investigated. The C3:0, C4:0, C6:0 and C10:0 ethanolamides neither affected basal Ca2+‐influx, nor the influx in response to a submaximal concentration of AEA (1 μM). In contrast, the C12:0, C17:0, C18:0 ethanolamides and the monounsaturated compound oleoylethanolamide (C18:1) greatly potentiated the response to AEA. Palmitoylethanolamide (C16:0) produced both a response per se and an augmentation of the response to AEA. Lauroylethanolamide (C12:0) produced a leftward shift in the dose‐response curve for AEA. EC50 values for AEA to produce Ca2+ influx into hVR1‐HEK293 cells were 1.8, 1.5, 1.1 and 0.22 μM in the presence of 0, 1, 3 and 10 μM lauroylethanolamide, respectively. Lauroylethanolamide did not affect the dose – response curves to capsaicin. Palmitoylethylamide was synthesized and found to be a mixed‐type inhibitor (Ki(slope) 4.1 μM, Ki(intercept) 66 μM) of [3H]‐AEA metabolism by rat brain membranes. The ‐amide, ‐ethylamide, ‐isopropylamide, ‐butylamide, ‐cyclohexamide and ‐trifluoromethyl ketone analogues of palmitoylethanolamide had little or no effect on the Ca2+ influx response to 1 μM AEA. There was no obvious relation between the abilities of the compounds to enhance the Ca2+ influx response to 1 μM AEA into hVR1‐HEK293 cells and to prevent the hydrolysis of AEA by rat brain membranes. It is concluded that although palmitoylethanolamide has entourage‐like effects at VR1 receptors expressed on hVR1‐HEK293 cells, other N‐acyl ethanolamines have even more dramatic potentiating effects. It is possible that they may play an important role under conditions where their synthesis is increased, such as in severe inflammation.

Platelet monoamine oxidase activity in sensation seekers

Personality traits, as assessed by personality inventories, and platelet monoamine oxidase activities have been compared in the following two groups of normal subjects: (1) subjects who either have mountaineering as an active hobby or an interest in mountaineering and (2) subjects not interested in mountaineering. The group of subjects interested in mountaineering were found to be sensation seekers, as assessed by the Zuckerman Sensation Seeking Scale, and to have significantly lower platelet monoamine oxidase activities.

Titration of human brain monoamine oxidase-A and-B by clorgyline andl-deprenil

SummaryThe interaction of clorgyline andl-deprenil with the-A and-B forms of human brain monoamine oxidase (MAO) has been studied. Both compounds inhibit cerebrocortical MAO in a manner consistent with a ‘suicide’ inactivation of the enzyme. The interaction of clorgyline with the-A form of the enzyme appears to take place almost entirely at specific binding sites, and the conditions required for this inhibitor to ‘titrate’ the concentrations of MAO-A have been elucidated.l-Deprenil has also been used to titrate the concentration of the-B form of MAO in cerebrocortical homogenates, but there is a considerable degree of non-specific binding of this compound. The two inhibitors have been used to titrate the concentrations of the two enzyme forms in frontal cortex homogenates from different age groups. There was a significantly higher MAO-B activity for the age range 73–95 years than for the age range 2–63 years. No significant differences between the two age groups were found for MAO-A. The activity of MAO-A in the samples correlated very well with the concentration of this enzyme form. Titration of the B-form of the enzyme withl-deprenil indicated an increased enzyme concentration with age, although other factors, such as the non-specific binding of this compound, could contribute to this effect.

The interaction between human platelet monoamine oxidase, its monoamine substrates and oxygen.

The interaction of human platelet MAO-B with three substrates (β-phenethylamine, tryptamine and benzylamine) has been investigated in an attempt to determine whether or not there exists heterogeneity of this enzyme form. Treatment with pargyline, thermal denaturation and 2-butanone affected the enzyme activity to the same degree, regardless of the amine substrate used. Mixed substrate experiments indicated that the substrates inhibited each other in a competitive manner with Ki values close to their Km values. The activity of MAO-B was increased in an uncompetitive manner as the oxygen concentration was raised. However, the degree of this increase was dependent upon the amine substrate used to assay for activity. These results are consistent for an enzyme with a single binding site for amine substrates and a possible multiplicity of binding sites for oxygen.

Transport of endocannabinoids across the plasma membrane and within the cell.

Endocannabinoids are readily accumulated from the extracellular space by cells. Although their uptake properties have the appearance of a process of facilitated diffusion, it is by no means clear as to whether there is a plasma membrane transporter dedicated to this task. Intracellular carrier proteins that shuttle the endocannabinoid anandamide from the plasma membrane to its intracellular targets such as the metabolic enzyme, fatty acid amide hydrolase, have been identified. These include proteins with other primary functions, such as fatty‐acid‐binding proteins and heat shock protein 70, and possibly a fatty acid amide hydrolase‐like anandamide transporter protein. Thus, anandamide uptake can be adequately described as a diffusion process across the plasma membrane followed by intracellular carrier‐mediated transport to effector molecules, catabolic enzymes and sequestration sites, although it is recognized that different cells are likely to utilize different mechanisms of endocannabinoid transport depending upon the utility of the endocannabinoid for the cell in question.

Lack of selectivity of URB602 for 2-oleoylglycerol compared to anandamide hydrolysis in vitro.

Two compounds, URB602 and URB754, have been reported in the literature to be selective inhibitors of monoacylglycerol lipase, although a recent study has questioned their ability to prevent 2‐arachidonoyl hydrolysis by brain homogenates and cerebellar membranes. In the present study, the ability of these compounds to inhibit monoacylglycerol lipase and fatty acid amide hydrolase has been reinvestigated.