Effects of Indole Alkaloids and Related Compounds on the Properties of Brain Microtubular Protein (original) (raw)
Related papers
Neurochemistry International, 1993
Al~traet--In the central nervous system, choline is an essential precursor of choline-containing phospholipids in neurons and glial cells and of acetylcholine in cholinergic neurons. In order to study choline transport and metabolism in the brain, we developed a comprehensive methodical procedure for the analysis of choline and its major metabolites which involves a separation step, selective hydrolysis and subsequent determination of free choline by HPLC and electrochemical detection. In the present paper, we report the levels of choline, acetylcholine, phosphocholine, glycerophosphocholine and choline-containing phospholipids in brain tissue, cerebrospinal fluid and blood plasma of the untreated rat. The levels of free choline in blood plasma (11.4 #M), CSF (6.7 #M) and brain intracellular space (64.0 pM) were sufficiently similar to be compatible with an exchange of choline between these compartments. In contrast, the intracellular levels of glycerophosphocholine (1.15 mM) and phosphocholine (0.59 mM) in the brain were considerably higher than their CSF concentrations of 2.83 and 1.70/~M, respectively. In blood plasma, glycerophosphocholine was present in a concentration of 4.58 #M while phosphocholine levels were very low or absent (< 0.1 #M). The levels of phosphatidylcholine and lyso-phosphatidylcholine were high in blood plasma (1267 and 268 #M) but very low in cerebrospinal fluid (< 10 pM). We concluded that the transport of free choline is the only likely mechanism which contributes to the supply of choline to the brain under physiological conditions.
Choline and Its Products Acetylcholine and Phosphatidylcholine
Handbook of Neurochemistry and Molecular Neurobiology, 2009
Choline, a quaternary amine obtained largely from the diet but also synthesized in the brain and, especially, liver, is an essential precursor of the neurotransmitter acetylcholine (ACh) and of the major membrane constituent phosphatidylcholine (PC). Plasma choline concentrations can vary over a fivefold range depending on the choline contents of the foods being digested. Since choline readily crosses the blood-brain barrier (BBB) through an unsaturated facilitated-diffusion system, these plasma changes can produce parallel changes in brain choline levels. In addition, since the enzymes that convert choline to ACh [choline acetyltransferase (ChAT)] and PC's precursor phosphocholine [choline kinase (CK)] are also poorly saturated with their choline substrate, increases in plasma choline can enhance the formation of ACh and phosphocholine, and the release of ACh. The subsequent conversion of phosphocholine to PC is increased if PC's other circulating precursors (uridine and omega-3 fatty acids) are provided. This leads to an increase in the levels of synaptic membrane within the brain. Choline is principally metabolized in the liver to betaine, which provides a source of methyl groups for the regeneration of methionine and S-adenosylmethionine.
Brain Research, 1973
As suggested by in vitro studies 7 brain tissue is unable to synthesize choline from its normal unmethylated precursors such as serine or monoaminoethanol. Similar conclusions could be drawn from in vivo experiments 1. An external supply of choline seems thus to be required for brain metabolism; in fact, this precursor is synthesized in the liver and probably transported by the plasma to the brain in a bound form (phosphatidylcholine)L However, several studies a,s,24 strongly suggest that free choline is the physiological precursor of acetylcholine (ACh) in brain. Indeed this molecule is taken up into brain synaptosomes by a carrier-mediated transport systemll,lZ, 21, apparently highly developed in cholinergic terminals 19. Such a dependence of neuronal tissue on external choline suggests that the availability of the precursor could be a limiting factor in ACh synthesis, or at least that the effect of drugs on choline uptake could be involved in some of their pharmacological actions.
The Journal of Neuroscience, 1985
to heat inactivation, differential loss of the enzyme forms in the Three fractions of choline 0-acetyltransferase (ChAT) (EC 2.3.1.6) were solubilized from a nerve ending fraction of rat forebrain using three sequential washes of an increasingly chaotrophic nature (100 mM sodium phosphate, pH 7.4; 500 mM NaCI; 2% Triton DN-65) as previously described (Benishin, C. G., and P. T. Carroll (1963) J. Neurochem. 41: 1030-1039). The molecular weights of the soluble (Nap) and membrane-bound fractions (NaCI and 2% Triton DN-65) of ChAT, following partial purification, were determined using either gel filtration on Sephadex G-200, G-100 Superfine, or sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by "Western blotting" and immunochemical visualization of ChAT with four different anti-ChAT monoclonal antibodies (Ab8, Ab9, 4D7, and lE6). Results obtained with gel filtration indicated that the NaP-and Triton DN-65solubilized fractions of ChAT had molecular weights in the range of 73,000 to 78,000, whereas the NaCI-solubilized fraction of ChAT had a molecular weight in the range of 230,000 to 240,000. Results obtained with SDS-PAGE and Western blotting indicated that all three fractions of ChAT were composed of the same nonidentical subunits. hippocampus following interruption of the septohippocampal pathway, and rate of development in neonatal brain (Benishin and Carroll, 1983, 1984). The objective of the present study was to determine whether these three fractions of ChAT also differed in molecular weight. To accomplish this objective, two procedures were used: (7) gel filtration in Sephadex G-100 Superfine and G-200, and (2) sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by "Western blotting" and immunochemical visualization of ChAT with four different monoclonal antibodies (Ab8, Ab9, 4D7, and lE6). The results indicated that the molecular weight of the native form of the high salt-soluble fraction of ChAT clearly exceeded the molecular weights of the sodium phosphate-and detergent-soluble fractions of ChAT. However, when the molecular weights of these ChAT fractions were determined on denaturing gels, none of these three fractions could be distinguished from each other. Instead, they all appeared to consist of the same nonidentical subunits. Materials and Methods Choline 0-acetyltransferase (EC 2.3.1.6; ChAT) catalyzes the formation of acetylcholine (ACh) in central cholinergic neurons. Although the majority of this enzyme is believed to be soluble and to exist in the cytoplasm of central cholinergic nerve terminals (Fonnum, 1968), some of it appears to be membrane bound (Smith and Carroll, 1980; Benishin and Carroll, 1981). Following hypoosmotic rupture of central cholinergic nerve terminals prepared from mouse or rat brain, soluble ChAT binds ionically to membranes and can be removed by repeated washes of the synaptosomal fraction with 100 mM sodium phosphate buffer (pH 7.4). Two membranebound fractions can then be solubilized by consecutive washes with 500 mM NaCl and 2% Triton DN-65 (Benishin and Carroll, 1983). The enzyme fractions removed by high salt and detergent differ from each other and from the soluble fraction in the following respects: pH profile, capacity to acetylate homocholine, sensitivity Bovine serum albumin (BSA), thyroglobulin, catalase, aldolase, ovalbumin, chymotrypslnogen A, blue dextran 2000, and Sephadex G-200 and G-100 Superfine were obtained from Pharmacia Fine Chemicals (Plscataway, NJ). EDTA, EGTA, phenylmethylsulfonyl fluoride (PMSF), pepstatin, soybean trypsin inhibitor, leupeptln, aprotinln, antipain dihydrochloride, benzamidine hydrochloride, benzethonium chlortde, choline bromide, eserine sulfate, acetyl-CoA, Triton DN-65, Nonidet P-40 (NP-40) and naive rat IgG were from Sigma Chemical Co. (St. Louis, MO). SDS low and high molecular weight protein standards and Bio-Beads SM.2 were from Bio-Rad Laboratories (Richmond, CA). Spectrafluor and [acetyl-1-?]acetyl-CoA (specific activity, 51 mCI/ mmol) were purchased from Amersham Corp. (Arlington Heights, IL). Hyamine hydroxide was obtained from Research Products International Corp.
Biosynthesis of Rat Brain Phosphatidylcholines from Intracerebrally Injected Choline
Journal of Neurochemistry, 1976
Ab~tract-[Me-~H]Choline was injected intracerebrally into male rats and the brains immediately removed by particular procedures at regular intervals over the first 1200 s. The incorporation of radioactivity into brain phosphorylcholine, CDP-choline and phosphatidylcholines was examined and quantitated, in order to investigate the relative roles of net synthesis and base-exchange reactions for choline incorporation into lipid. The molecular subspecies of phosphatidylcholines were also examined after isotope administration. Phosphorylcholine, CDP-choline and phosphatidylcholines all became labelled as early as 5 s after the administration of labelled choline. The time course of incorporation of choline into brain lipid is biphasic with two flex points at about 20 and 120 s from the injection. The specific radioactivity of different phosphatidylcholines appears to be different at early and later intervals from injection. The suggestion is made that the base-exchange pathway for choline incorporation into lipid might be operative in viw in early periods after administration. GAIT1 A., DE MEDIO G. E., BRUNETTI M., AMADUCCI L.
Oral Cytidine 5'Diphosphate Choline Administration to Rats Increases Brain Phospholipid Levels
Annals of The New York Academy of Sciences, 1993
Exogenous cytidine 5′diphosphocholine (CDF-choline) is completely metabolized to circulating cytidine and choline. Both compounds enter the brain and can be used in phosphatidylcholine (PC) synthesis via the Kennedy (CDP-choline) cycle. We administered oral CDP-choline to 12 month-old rats (500 mg/kg/day) for 21, 42, or 90 days to determine whether this treatment would alter brain levels of PC and the other structural phospholipids, phosphatidylserine (PS) and phosphatidylethanolamine (PE). After 42 days, brain PC levels increased significantly (p < 0.01) by 23.3%; after 90 days PC increased by 30% (p < 0.01), PS by 37.2% (p < 0.01), and PE by 13% (not significant). The ratios of each of the phospholipids to total membrane phospholipids were unchanged. These data demonstrate that repeated oral CDP-choline administration can increase the amounts of phospholipids in brain membranes, thus providing a rationale for using this compound in brain diseases that damage neurons.
Choline Uptake by Cerebral Capillary Endothelial Cells in Culture
Journal of Neurochemistry, 1990
A passage of choline from blood to brain and vice versa has been demonstrated in vivo. Because of the presence of the blood-brain barrier, such passage takes place necessarily through endothelial cells. To get a better understanding of this phenomenon, the choline transport properties of cerebral capillary endothelial cells have been studied in vitro. Bovine endothelial cells in culture were able to incorporate [3H]choline by a carrier-mediated mechanism. Nonlinear regression analysis of the uptake curves suggested the presence of two transport components in cells preincubated in the absence of choline. One component showed a K , of 7.59 k 0.8 pM and a maximum capacity of 142.7 k 9.4 pmol/2 min/mg of protein, and the other one was not saturable within the concentration range used (1-100 pM). When cells were preincubated in the presence of choline, a single saturable component was observed with a K,,, of 18.5 * 0.6 p M and a maximum capacity of 452.4 k 42 pmol/2 min/mg of protein.
Brain Research, 1985
cerebral cortex cup technique --blood-brain barrier --physostigmine --penicillin Effiux of choline from the rat cerebral cortex in vivo was investigated using the cup technique. After removal of the dura mater, the cup was placed on the cortex. Transmission and scanning electron microscopy revealed that the cortex was separated from the cup solution (100-300 gl) by basal lamina, pia mater, arachnoid (with discrete defects) and remainders of the snbdural neurothelium. Two kinds of experiments were carried out to determine: (1) efflux of unlabelled choline into the cup solution; and (2) transioeation of radioactivity from the plasma into the cup solution (via blood-brain barrier and leptomeningeal layers) during i.v. infusion of [SH]choline or [14C]inulin. The former process was highly temperature-sensitive in contrast to the latter. Penicillin-G-sodium, which is known to damage the blood-brain barrier, was added to the cup solution, enhanced effiux of unlabelled choline, and caused a 5-fold increase in the rates of translocation of radioactivity during infusion of either labelled choline or inulin. In contrast, physostigmine (3 x 10 -4 M, added to cup solution) failed to enhance 3H-translocation. but markedly facilitated the efflux of unlabelled choline, this effect was highly temperature-sensitive and was blocked by atropine. It is concluded that activation of muscarinic receptors enhanced the choline efflux from cortical tissue. This effect was caused by cellular mobilization of choline presumably through an action on the metabolism of phosphatidylchotine. The effect was not due to alterations in the translocation of choline from the plasma to the cup solution, i.e. through permeability changes in the blood-brain barrier and in the teptomeningeal 'barrier'. The cup technique appears to be a useful technique for studying alterations in the blood-brain barrier in vivo.
Biosynthesis of Rat Brain Phosphatidylcholines from Intracerebrally Injected CHOLINE1
Journal of Neurochemistry, 1976
Ab~tract-[Me-~H]Choline was injected intracerebrally into male rats and the brains immediately removed by particular procedures at regular intervals over the first 1200 s. The incorporation of radioactivity into brain phosphorylcholine, CDP-choline and phosphatidylcholines was examined and quantitated, in order to investigate the relative roles of net synthesis and base-exchange reactions for choline incorporation into lipid. The molecular subspecies of phosphatidylcholines were also examined after isotope administration. Phosphorylcholine, CDP-choline and phosphatidylcholines all became labelled as early as 5 s after the administration of labelled choline. The time course of incorporation of choline into brain lipid is biphasic with two flex points at about 20 and 120 s from the injection. The specific radioactivity of different phosphatidylcholines appears to be different at early and later intervals from injection. The suggestion is made that the base-exchange pathway for choline incorporation into lipid might be operative in viw in early periods after administration. ' This work has been aided by a research grant from the Consiglio Nazionale delle Richerche, Rome (Contract n. 74.00259).
Journal of Neurochemistry, 2002
We examined the effects of orally administered 5'-cytidinediphosphocholine (CDP-choline) on arterial plasma choline and cytidine levels and on brain phospholipid composition in rats. Animals receiving a single oral dose of 100, 250, or 500 mg/kg showed peak plasma choline levels 6-8 h after drug administration (from 12 1 to 17 ± 2, 19 ± 2, and 24 ± 2 pM, respectively). The area under the plasma choline curve at >14 IiM, i.e ., at a concentration that induces a net influx of choline into the brain, was significantly correlated with CDP-choline dose. In rats receiving 500 mg/kg this area was 2 .3 times that of animals consuming 250 mg/kg, which in turn was 1 .8 times that of rats receiving 100 mg/kg. Plasma cytidine concentrations increased 5.4, 6.5, and 15 .1 times baseline levels, respectively, 8 h after each of the three doses. When the oral CDP-choline treatment was prolonged for 42 and 90 days, brain phosphatidylcholine concentrations increased significantly (by 22-25% ; p < 0.05) in rats consuming 500 mg/kg/day. Brain phosphatidylethanolamine and phosphatidylserine concentrations also increased significantly under some experimental conditions ; levels of other phospholipids were unchanged.