Brain Constituents Research Articles

Inhibitors of arachidonoyl ethanolamide hydrolysis.

Arachidonoyl ethanolamide (anandamide) is a naturally occurring brain constituent that binds to a specific brain cannabinoid receptor (CBR1). An amidase activity (anandamide amidase) in membrane fractions of brain and in cultured neuroblastoma cells rapidly degrades anandamide to arachidonic acid (Deutsch, D. G., and Chin, S. (1993) Biochem. Pharmacol. 46, 791-796). In the current study, analogs of anandamide representing three classes of putative transition-state inhibitor (trifluoromethyl ketones, alpha-keto esters, and alpha-keto amides) were synthesized and tested as inhibitors of anandamide hydrolysis in vitro and as ligands for CBR1. The trifluoromethyl ketones and alpha-keto esters showed nearly 100% inhibition of anandamide hydrolysis in vitro at 7.5 microM inhibitor and 27.7 microM anandamide. Arachidonyl trifluoromethyl ketone was the only synthetic compound in the series of fatty acid derivatives able to displace [3H]CP-55940 binding to CBR1 with a Ki of 0.65 microM. It was also the most effective inhibitor in intact neuroblastoma cells, leading to a 12-fold increase of cellular anandamide levels at 12 microM. From the action of these inhibitors on this hydrolytic enzyme, it seems likely that anandamide is cleaved by a mechanism that involves an active-site serine hydroxyl group. These inhibitors may serve as useful tools to elucidate the role anandamide plays in vivo.

Inhibition of glutamate dehydrogenase in brain mitochondria and synaptosomes by Mg2+ and polyamines: a possible cause for its low in vivo activity.

Magnesium and the polyamines putrescine, spermidine, and spermine inhibited the activity of glutamate dehydrogenase in permeabilized rat brain mitochondria in a concentration-dependent manner. The inhibitory effect was observed on both the reductive amination of 2-oxoglutarate and oxidative deamination of glutamate, as well as in the presence and absence of ADP and leucine, the allosteric activators of the enzyme. Kinetic studies at various concentrations of substrates showed that inhibition by magnesium and spermine was very pronounced at 2-oxoglutarate concentrations less than 0.5 mM and NADH levels less than 0.08 mM. The presence of the former compounds also accentuated the inhibitory effect of high concentrations of 2-oxoglutarate (> 2.0 mM) and NADH (> 0.32 mM). Addition of magnesium and spermine to suspensions of synaptosomes decreased the amount of ammonia produced from glutamate. It is suggested that polyamines and magnesium, normal constituents of mammalian brain, are responsible, at least in part, for the low glutamate dehydrogenase activity in vivo.

Effect of the brain constituent anandamide, a cannabinoid receptor agonist, on the hypothalamo-pituitary-adrenal axis in the rat.

Anandamide (arachidonylethanolamide), an endogenous ligand of the cannabinoid receptor, was recently isolated from porcine brain. We report here for the first time on the effect of this ligand on the hypothalamo-pituitary adrenal (HPA) axis in comparison to that of the plant cannabinoid delta 9-tetrahydrocannabinol (THC). Intracerebroventricular injection of anandamide or THC (50 or 150 micrograms/rat) increased significantly the serum levels of ACTH and corticosterone in a dose-dependent manner and caused a pronounced depletion of CRF-41 in the median eminence. These data suggest that anandamide parallels THC in activating the HPA axis via mediation of a central mechanism which involves the secretion of CRF-41.

The Effect of Cannabidiol on Mouse Hepatic Microsomal Cytochrome P450-Dependent Anandamide Metabolism

Anandamide (arachidonylethanolamide) has been identified as a brain constituent that selectively binds to the cannabinoid receptor and possesses cannabimimetic activity. Cytochromes P450 catalyze the oxidation of arachidonic acid to several metabolites possessing very potent pharmacological activity. We examined whether P450 would also metabolize anandamide, and whether cannabidiol (a cannabinoid which inactivates several P450s) would affect this metabolism. Mouse hepatic P450s were found to metabolize anandamide to at least 10 different metabolites, four of which were characterized by mass spectrometry. Cannabidiol selectively inhibited the formation of two of these four anandamide metabolites. The significance of anandamide metabolism remains to be explored.

Enzymatic synthesis and degradation of anandamide, a cannabinoid receptor agonist

Enzymatic activities have been identified which catalyze both the hydrolysis and synthesis of arachidonylethanolamide (anandamide). Anandamide was taken up by neuroblastoma and glioma cells in culture, but it did not accumulate since it was rapidly degraded by an amidase activity that resided mainly in the membrane fractions. This amidase activity was expressed in brain and the majority of cells and tissues tested. Phenylmethylsulfonyl fluoride (PMSF) was found to be a potent inhibitor of this amidase. A catalytic activity for the biosynthesis of anandamide from ethanolamine and arachidonic acid was readily apparent in incubations of rat brain homogenates. The stability of anandamide in serum and its rapid breakdown in cells and tissues are consistent with the observation that it is active when administered systemically, and its duration of action will be regulated by its rate of degradation in cells. The psychoactive marijuana plant-derived cannabinoid, Δ 9-tetrahydrocannabinol, and numerous synthetic derivatives have been shown to bind to a specific brain receptor [1,2,3,4]. Arachidonylethanolamide has been identified recently as a naturally occurring brain constituent that binds to this cannabinoid receptor [5]. Furthermore, using a series of behavioral tests to evaluate cannabinoid analogs [6], anandamide has been shown to be a cannabinoid receptor agonist exhibiting pharmacological activity in mice parallel to that of other psychotropic cannabinoids [7]. In this study, the uptake and breakdown of anandamide in neuroblastoma and glioma cell cultures were characterized. Subcellular fractions of these cultured cells and crude homogenates of other cultured cells and of rat tissues were assayed for enzymatic breakdown of arachidonylethanolamide to arachidonic acid. It was observed that phenylmethylsulfonyl flouride (PMSF) was a potent inhibitor of the breakdown activity (anandamide amidase). Employing ethanolamine and arachidonic acid as substrates, a synthetic activity (anandamide synthase) was readily demonstrated in crude homogenates of rat brain.

Erratum: Pot, Heroin Unlock New Areas for Neuroscience

In the Research News article "Pot, heroin unlock new areas for neuroscience" by Marcia Barinaga (18 Dec., p. 1882), the diagram of the molecule anandamide on page 1883 was incorrect. The correct structure appears on page 1948 of the same issue, in figure 1A of the report "Isolation and structure of a brain constituent that binds to the cannabinoid receptor" by W. A. Devane et al. (p. 1946).

Big Physics and New Ideas

In the Research News article "Pot, heroin unlock new areas for neuroscience" by Marcia Barinaga (18 Dec., p. 1882), the diagram of the molecule anandamide on page 1883 was incorrect. The correct structure appears on page 1948 of the same issue, in figure 1A of the report "Isolation and structure of a brain constituent that binds to the cannabinoid receptor" by W. A. Devane et al. (p. 1946).

Erratum: Pot, Heroin Unlock New Areas for Neuroscience

In the Research News article "Pot, heroin unlock new areas for neuroscience" by Marcia Barinaga (18 Dec., p. 1882), the diagram of the molecule anandamide on page 1883 was incorrect. The correct structure appears on page 1948 of the same issue, in figure 1A of the report "Isolation and structure of a brain constituent that binds to the cannabinoid receptor" by W. A. Devane et al . (p. 1946).

Pharmacological activity of the cannabinoid receptor agonist, anandamide, a brain constituent

Anandamide (arachidonylethanolamide) is a brain constituent which binds to the cannabinoid receptor. We now report the first in vivo examination of this ligand. Anandamide administered i.p. in mice, caused lowering of activity in an immobility and in an open field test, and produced hypothermia and analgesia. These effects parallel those caused by psychotropic cannabinoids.

Steroids and their conjugates in the mammalian brain.

Five steroids--3 beta-hydroxypregn-5-en-20-one (pregnenolone; P), 3 beta-hydroxy-5 alpha-pregnan-20-one (3 beta-AP), 3 alpha-hydroxy-5 alpha-pregnan-20-one (3 alpha-AP), 3 beta-hydroxyandrost-5-en-17-one (dehydroepiandrosterone; D), and 3 beta-hydroxy-5 alpha-androstan-17-one (EpiA)--were extracted from the brains of adult male rats, rabbits, and dogs. The steroids exist in this organ as unconjugated compounds and as sulfates, lipoidal esters, and sulfolipids. The techniques for separating these four classes of steroids from each other and for separating the five steroids from each other are described. In all cases, the steroids were identified by their retention time (Rt) on HPLC, their Rt by gas chromatography, and by selected ion monitoring of their mass spectra. The latter were also used for quantification. In their reaction toward organic bases, the sulfolipid conjugates resemble previously described sulfolipids of cholesterol and sitosterol. These conjugates are relatively abundant in brain, particularly those of P and D, and this suggests that, in the search for the physiological significance of these brain constituents, these conjugates warrant attention.

Isolation and structure of a brain constituent that binds to the cannabinoid receptor.

Arachidonylethanolamide, an arachidonic acid derivative in porcine brain, was identified in a screen for endogenous ligands for the cannabinoid receptor. The structure of this compound, which has been named "anandamide," was determined by mass spectrometry and nuclear magnetic resonance spectroscopy and was confirmed by synthesis. Anandamide inhibited the specific binding of a radiolabeled cannabinoid probe to synaptosomal membranes in a manner typical of competitive ligands and produced a concentration-dependent inhibition of the electrically evoked twitch response to the mouse vas deferens, a characteristic effect of psychotropic cannabinoids. These properties suggest that anandamide may function as a natural ligand for the cannabinoid receptor.

Polyamines potentiate responses of N-methyl-D-aspartate receptors expressed in xenopus oocytes.

Glutamate, the major excitatory neurotransmitter in the central nervous system, activates at least three types of channel-forming receptors defined by the selective agonists N-methyl-D-aspartate (NMDA), kainate, and quisqualate [or more selectively by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)]. Activation of the NMDA receptor requires glycine as well as NMDA or glutamate. Recent studies have provided evidence that certain polyamines potentiate the binding by NMDA receptors of glycine and the open channel blocker MK-801. To determine whether polyamines alter channel opening, we examined their effects on rat brain glutamate receptors expressed in Xenopus oocytes. Our results demonstrate that spermine potentiates the response of the NMDA receptor but has no effect on responses to kainate and quisqualate. Furthermore, spermine increases the maximum response to NMDA and glycine and acts, at least in part, by increasing the apparent affinity of the NMDA receptor/channel complex for glycine. The present findings and the fact that polyamines are a natural constituent of brain suggest that polyamines may play a role in the regulation of glutamatergic transmission.

Proton magnetic resonance spectroscopy in multiple sclerosis

Regional in vivo proton magnetic resonance spectroscopy provides quantitative data on selected chemical constituents of brain. We imaged 16 volunteers with clinically definite multiple sclerosis on a 1.5 tesla magnetic resonance scanner to define plaque-containing volumes of interest, and obtained localized water-suppressed proton spectra using a stimulated echo sequence. Twenty-five of 40 plaque-containing regions provided spectra of adequate quality. Of these, 8 spectra from 6 subjects were consistent with the presence of cholesterol or fatty acids; the remainder were similar to those obtained from white matter of normal volunteers. This early experience with regional proton spectroscopy suggests that individual plaques are distinct. These differences likely reflect dynamic stages of the evolution of the demyelinative process not previously accessible to in vivo investigation.

Clipsin, a chymotrypsin-like protease in rat brain which is irreversibly inhibited by alpha-1-antichymotrypsin.

The protease inhibitor alpha-1-antichymotrypsin, which binds to chymotrypsin-like enzymes in a sodium dodecyl sulfate-resistant manner, has been shown recently to be both a normal constituent of brain and an integral component of the neuritic plaques that form in Down's syndrome and Alzheimer's disease. We have now identified in rat brain a Mr 25,000 alpha-1-antichymotrypsin-binding protein classified as a chymotrypsin-like protease by its inhibitor profile and substrate specificity. Release of 125I-labeled breakdown products from bands containing the protease in substrate-linked polyacrylamide gels was examined in parallel with hydrolysis of tetrapeptide chromogenic substrates in vitro to establish conditions under which the Mr 25,000 protease was the only activity being measured in vitro. The protease was completely membrane associated but was extractable using 1 M MgCl2; prior extraction of detergent- and low ionic strength-soluble proteins from membranes was used to increase its specific activity. The formation of sodium dodecyl sulfate-resistant bonds between human alpha-1-antichymotrypsin and the protease (kassoc = 2.9 X 10(6) M-1 s-1) was used to titrate the concentration of free protease solubilized from membranes. The protease cleaved both succinyl-Ala-Ala-Pro-Phe-p-nitroanilide, and methoxy-succinyl-Ala-Ala-Pro-Met-p-nitroanilide, the latter being of interest because cleavage after a methionine residue is predicted to generate the amino terminus of the neuritic plaque component beta-amyloid from its precursor protein. In fact, the solubilized protease degraded 90% of membrane-associated beta-amyloid precursor protein detected by Western blot analysis. The protease was kinetically distinct from both chymotrypsin and cathepsin G in direct comparisons and did not match kinetic values published for the rat mast cell proteases against comparable substrates; we therefore refer to the protease with the descriptive acronym clipsin (for chymotrypsin-like protease). Proteases similar to and potentially identical to clipsin were detected by enzymography in other organs from rat (most notably spleen and adult lung). The enzyme in brain was distinguished by a narrow window of elevated activity surrounding postnatal day 5, which was 12-14-fold higher than levels in day 1 or adult brain. Because independent lines of evidence suggest that a brain chymotrypsin-like protease may be involved in the etiology of Down's syndrome and Alzheimer's disease, clipsin is discussed as a candidate for such a role.

Identification of the plasma membrane proteolipid protein as a constituent of brain coated vesicles and synaptic plasma membrane.

We have analyzed brain coated vesicles and synaptic plasma membrane for the presence of the plasma membrane proteolipid protein. Coated vesicles were isolated from calf brain gray matter with a final purification on Sephacryl S-1000 and reisolated twice by chromatography to ensure homogeneity. Fractions were analyzed by gel electrophoresis, immunoblotting for clathrin heavy chain, and by electron microscopy. Using an immunoblotting assay we were able to demonstrate the presence of the plasma membrane proteolipid protein in these coated vesicles at a significant level (i.e., approximately 1% of the bilayer protein of these vesicles). Reisolation of coated vesicles did not diminish the concentration of the protein in this fraction. Removal of the clathrin coat proteins or exposure of the coated vesicles to 0.1 M Na2CO3 showed that the plasma membrane proteolipid protein is not removed during uncoating and lysis but is intrinsic to the membrane bilayer of these vesicles. These studies demonstrate that plasma membrane proteolipid protein represents a significant amount of the bilayer protein of coated vesicles, suggesting that these vesicles may be a transport vehicle for the intracellular movement of the plasma membrane proteolipid protein. Isolation of synaptic plasma membranes proteolipid adult rat brain and estimation of the plasma membrane proteolipid protein content using the immunoblotting method confirmed earlier studies that show this protein is present in this membrane fraction at high levels as well (approximately 1-2%). The level of this protein in the synaptic plasma membrane suggests that the synaptic plasma membrane is one major site to which these vesicles may be targeted or from which the protein is being retrieved.

Complex ganglioside expression and tetanus toxin binding by PC12 pheochromocytoma cells.

Ganglioside expression and tetanus toxin binding were studied in the rat pheochromocytoma cell line PC12. Seven ganglioside species were readily detected in extracts of PC12 cells; two were identified as tri- and tetrasialogangliosides, which are common brain constituents but unusual components of neuronal cell lines. Carbohydrate composition, acid and enzyme hydrolyses, and mass spectral analysis revealed that the major species is GT 1b, a predominant mammalian brain ganglioside previously reported to support high affinity tetanus toxin binding (Rogers, T. B., and Snyder, S. H. (1981) J. Biol. Chem. 256, 2402-2407). Direct binding of 125I-tetanus toxin to PC12 gangliosides on TLC plates revealed selective binding to the tri- and tetrasialogangliosides. Radioiodinated toxin also bound with high affinity to intact PC12 cells or their isolated membranes. The binding affinity (Kd = 1.25 nM), density of receptors (Bmax = 238 pmol/mg of membrane protein), and dependence on pH, ionic strength, and temperature were similar to those previously reported for toxin binding to rat brain synaptic membranes. Differentiation of PC12 cells caused an increase in expression of the tri- and tetrasialogangliosides and a closely matched increase in tetanus toxin binding to cell membranes. These data provide evidence that complex gangliosides may act as tetanus toxin receptors, and demonstrate the utility of the PC12 cell line for studies of tetanus toxicity and complex ganglioside expression.

Chapter 37 Blood-brain and blood-cerebrospinal fluid alterations following neural transplantation

Publisher Summary The advent of a foreign graft can alter certain vascular properties and constituents of the host brain—namely, the blood-brain (BBB) or blood-cerebrospinal fluid (BCB) barriers. This chapter describes and demonstrates the changes in vascular permeability histochemically after grafting of fetal central nervous system (CNS) tissue or adrenal medullary tissue. The chapter also presents data concerning alterations in the BCB utilizing the technique of high-performance immunoaffinity chromatography (HPIAC) of CSF samples, and focuses on the vascular process in brain grafts and the effects of such neovascularization on the barrier properties in both host and graft. The transplantation of fetal CNS is quite different from autonomic tissue transplants, both physiologically and developmentally. When fetal brain is placed into host brain, particularly at brain surfaces where pial vessels have BBB properties, there appears to be a phenotypic change in certain endothelial cell properties following the neovascularization process. Entry of the potentially neuroactive compounds from the blood into the extracellular fluid (ECF) or CSF could change the host brain's controlled fluid environment, and it would seem likely that endogenous blood borne compounds also have access to the brain. However, how graft-produced products enter into the brain remains to be determined.

N-Acylethanolamine phospholipid metabolism in normal and ischemic rat brain

N-Acetylethanolamine phospholipids accumulate in rat brain during post-decapitative ischemia. Small amounts of these phospholipids consisting primarily of diacyl and alkenylacyl species can be detected within 15 min of ischemia and they increase linearly for 60 min. This ischemia-induced synthesis is more pronounced in developing rat brain (approx. 5.0 nmol/h per μmol lipid P) than in adult brain (0.4 nmol). Pulse labeling experiments with subcellular preparations of 10-day-old rat brain indicate a precursor-product relationship between ethanolamine phospholipids and their N-acyl analogs. N-Acylation of endogenous substrates occurs with both microsomes and mitochondria, exhibits a pH optimum of 10 and requires 1 mM Ca 2+ for maximal (0.2 mM Ca 2+ for half maximal) activity. Cell-free preparations of both developing and adult rat brain contain a phosphodiesterase which hydrolyzes N-acylphosphatidylethanolamine to phosphatidic acid and N-acylethanolamine. The latter is further hydrolyzed to fatty acid and ethanolamine by an amidohydrolase. [1- 3H]Ethanolamine, injected intracerebrally or intraperitoneally into 13- and 18-day-old rats, is incorporated into brain ethanolamine phospholipids. Since small amounts of radioactivity are also associated with N-acylethanolamine phospholipids 5 and 24 h after injection of the substrate, it appears that these phospholipids may occur at a very low level as a natural lipid constituent of rat brain.

P2B0104) - Pharmacological studies on alicyclic amines and amino acids. XXXXVIII: Microelectrophoretic study of 5-hydroxy pipecolic acid and 4-hydroxy proline, novel brain constituents in rat brain.

P2B0104) - Pharmacological studies on alicyclic amines and amino acids. XXXXVIII: Microelectrophoretic study of 5-hydroxy pipecolic acid and 4-hydroxy proline, novel brain constituents in rat brain.

Neuropeptides in CSF and post-mortem brain tissue of normal controls, schizophrenics and huntington's choreics

Manberg, Paul, J., Charles B. Memeroff, Garth Bissette, Eric Widerl̈ov, William W. Youngblood, John S. Kizer, and Arthur J. Prange, Jr. Prog. Neuro-Psychopharmacol. & Biol. Psychiat. 1985- 9 (1): 97–108. 1. 1. Studies describing the CNS distribution of neuropeptides can provide important new insights concerning their possible physiological functions. 2. 2. The rational for studying human post-mortem tissue, as well as some methodological constraints, are reviewed. 3. 3. The localization of NT in normal human brain is presented. 4. 4. Concentrations of NT, TRH, and SRIF were determined in brain tissue from normal controls and patients with schizophrenia or Huntington's chorea. Specific alterations in the levels of these neuropeptides were found in each disease. 5. 5. Appreciable quantities of NT immunoreactivity are present in human CSF. Sub-normal CSF-NT levels were found in a sub-group of unmedicated schizophrenics but were elevated back to normal concentrations following neuroleptic treatment. 6. 6. Although the pathophysiological significance of these findings is unclear, they nevertheless indicate that neuropeptides are important brain constituents which deserve further study.