Distribution in brain and retina of four enzymes of acetyl CoA synthesis in relation to choline acetyl transferase and acetylcholine esterase

Eleven regions of mouse brain and twelve layers of monkey retina were assayed for choline acetyl transferase (ChAT), acetylcholine esterase (AChE), and 4 enzymes that synthesize acetyl CoA. The purpose was to seek evidence concerning the source of acetyl CoA for acetylcholine generation. In brain ATP citrate lyase was strongly correlated with ChAT as well as AChE (r=0.914 in both cases). Weak, but statistically significant correlation, was observed between ChAT and both cytoplasmic and mitochondrial thiolase, whereas there was a significant negative correlation between ChAT and acetyl thiokinase. In retina ChAT was essentially limited to the inner plexiform and ganglion cell layers, whereas substantial AChE activity extended as well into inner nuclear, outer plexiform and fiber layers, but no further. ATP citrate lyase activity was also highest in the inner four retinal layers, but was not strongly correlated with either ChAT or AChE (r=0.724 and 0.761, respectively). Correlation between ChAT and acetyl thiokinase was at least as strong (r=0.757), and in the six inner layers of retina, the correlation between ChAT and acetylthiokinase was very strong (r=0.932).Special issue dedicated to Dr. Lawrence Austin     

Subcellular localization of enzymes oxidizing citrate in the rat brain

¹RSA was calculated as the ratio: percentage of total recovered activity found in fraction to percentage of total protein found in the same fraction.     

Regulation of the activity of choline acetyl transferase by lipoic acid

The observations reported in this article demonstrate that lipoic acid strongly influences the activity of a purified preparation of choline acetyl transferase. The reduced form, dihydrolipoic acid, is a powerful activator of the enzyme while lipoic acid itself has an inhibitory effect and counteracts the stimulatory effect of dihydrolipoic acid. It is proposed that dihydrolipoic acid serves an essential function in the action of this enzyme and that the ratio of reduced to oxidized lipoic acid in the cell may play an important role in the regulation of the activity of the enzyme. The implications of these findings for cell function and acetyl choline formation are discussed.Affiliation     

The source of choline for acetylcholine synthesis in brain

More is known about the synthesis and metabolism of acetylcholine (ACh) than other choline (Ch) containing compounds in the brain in spite of the fact that ACh represents only a small fraction of the total Ch esters. This review will attempt to summarize the evidence for the source of Ch in the brain and its relation to the turnover of ACh. Ch is a precursor not only for ACh but also for phosphoryl Ch and phospholipids. It appears that in the rat a bound form of Ch in the brain can produce free Ch which can leave the brain, be converted to ACh or be reutilized for phospholipid synthesis. There is evidence that one of the sources of free Ch that is utilized for ACh synthesis is outside the cholinergic nerve terminal.     

On the catalytic mechanism of human ATP citrate lyase

ATP citrate lyase (ACL) catalyzes an ATP-dependent biosynthetic reaction which produces acetyl-coenzyme A and oxaloacetate from citrate and coenzyme A (CoA). Studies were performed with recombinant human ACL to ascertain the nature of the catalytic phosphorylation that initiates the ACL reaction and the identity of the active site residues involved. Inactivation of ACL by treatment with diethylpyrocarbonate suggested the catalytic role of an active site histidine (i.e., His760), which was proposed to form a phosphohistidine species during catalysis. The pH-dependence of the pre-steady-state phosphorylation of ACL with [γ-(33)P]-ATP revealed an ionizable group with a pK(a) value of ~7.5, which must be unprotonated for the catalytic phosphorylation of ACL to occur. Mutagenesis of His760 to an alanine results in inactivation of the biosynthetic reaction of ACL, in good agreement with the involvement of a catalytic histidine. The nature of the formation of the phospho-ACL was further investigated by positional isotope exchange using [γ-(18)O(4)]-ATP. The β,γ-bridge to nonbridge positional isotope exchange rate of [γ-(18)O(4)]-ATP achieved its maximal rate of 14 s(-1) in the absence of citrate and CoA. This rate decreased to 5 s(-1) when citrate was added, and was found to be 10 s(-1) when both citrate and CoA were present. The rapid positional isotope exchange rates indicated the presence of one or more catalytically relevant, highly reversible phosphorylated intermediates. Steady-state measurements in the absence of citrate and CoA showed that MgADP was produced by both wild type and H760A forms of ACL, with rates at three magnitudes lower than that of k(cat) for the full biosynthetic reaction. The ATPase activity of ACL, along with the small yet significant positional isotope exchange rate observed in H760A mutant ACL (~150 fold less than wild type), collectively suggested the presence of a second, albeit unproductive, phosphoryl transfer in ACL. Mathematical analysis and computational simulation suggested that the desorption of MgADP at a rate of ~7 s(-1) was the rate-limiting step in the biosynthesis of AcCoA and oxaloacetate.     

Regulation of citrate metabolism and acetycholine synthesis by Ca in rat brain synaptosomes

The relationships between pyruvate and derived citrate metabolism and acetylcholine (ACh) synthesis in synaptosomes were examined. In the presence of 30 mM KCl, 0.1 mM Ca²⁺ caused 31 and 63% inhibition of pyruvate utilization and citrate accumulation, respectively. Verapamil and EGTA (0.5 mM) brought about no change in pyruvate consumption but increased rate of citrate accumulation, and overcame inhibitory effect of Ca²⁺. The rates of citrate accumulation in the presence of verapamil or EGTA were three to six times, respectively, higher than those in the presence of Ca²⁺. (−) Hydroxycitrate increased rate of citrate accumulation under all experimental conditions. The value of this activation appeared to be stable (0.20–0.28 nmol/min/mg of protein) and independent of changes in the basic rate of citrate accumulation. Ca²⁺ caused no significant changes in [¹⁴C]ACh synthesis, but it inhibited ¹⁴CO₂ production by synaptosomes. These activities were inhibited by verapamil by 33 and 60%, respectively. Ca²⁺ did not modify these effects of the drug. On the other hand, (−)hydroxycitrate resulted in 22 and 29% inhibition of [¹⁴C]ACh synthesis in Ca²⁺ free and Ca²⁺ supplemented medium, respectively. These data indicated that rates of acetyl-CoA synthesis in synaptoplasm, via ATP-citrate lyase and probably by another pathways are independent of Ca-evoked changes in pyruvate oxidation and citrate supply from intraterminal mitochondria. This property might play a significant role in maintenance of stable level of ACh in active cholinergic nerve endings.     

On the mechanism of citrate inhibition of ceruloplasmin ferroxidase activity

Ceruloplasmin is a plasma protein, which oxidizes ferrous ions in a catalytic manner. It is considered to function as a ferroxidasein vivo. Citrate was found to inhibit the reaction. The ceruloplasmin catalyzed oxidation ofp-phenylenediamines, however, was not affected by citrate. The inhibitory effect is proposed to be due to formation of Fe²⁺-citrate, which does not react with ceruloplasmin. The stability constant for the Fe²⁺-citrate complex estimated from the present inhibition study is in good agreement with previously published data.     

Inhibition of the rat blood–brain barrier choline transporter by manganese chloride

Choline transport has been characterized by multiple mechanisms including the blood-brain barrier (BBB), and high- and low-affinity systems. Each mechanism has unique locations and characteristics yet retain some similarities. Previous studies have demonstrated cationic competition by monovalent cations at the BBB and cation divalent manganese in the high-affinity system. To evaluate the effects of divalent manganese inhibition as well as other cationic metals at the BBB choline transporter, brain choline uptake was evaluated in the presence of certain metals of interest in Fischer-344 rats using the in situ brain perfusion technique. Brain choline uptake was inhibited in the presence of Cd(2+) (73 +/- 2%) and Mn(2+) (44 +/- 6%), whereas no inhibition was observed with Cu(2+) and Al(3+). Furthermore, it was found that manganese caused a reduction in brain choline uptake and significant regional choline uptake inhibition in the frontal and parietal cortex, the hippocampus and the caudate putamen (45 +/- 3%, 68 +/- 18%, 58 +/- 9% and 46 +/- 15%, respectively). These results suggest that choline uptake into the CNS can be inhibited by divalent cationic metals and monovalent cations. In addition, the choline transporter may be a means by which manganese enters the brain.     

Formation of unesterified choline by rat brain

Two preparations of rat brain (ischemic intact brain and homogenized whole brain) formed large amounts of unesterified (free) choline when incubated at 37 degrees C. The accumulation of choline was inhibited by microwave irradiation of brain, or by heating of brain to 50 degrees C, and was maximal at 37 degrees C at pH 7.4-8.5. Choline formation was only observed in subcellular fractions of brain that contained membranes. In homogenates of brain, choline accumulated at a rate exceeding 10 nmol/mg protein per h. There was a significant decrease in brain phosphatidylcholine concentration (of 50 nmol/mg protein) during incubation for 1 h at 37 degrees C. Concentrations of phosphocholine rose (by 2.3 nmol/mg protein), and concentrations of glycerophosphocholine and sphingomyelin did not change during this period. We used radiolabeled phospholipids to trace the fate of phosphatidylcholine and sphingomyelin during incubations of homogenates of brain. Phosphatidylcholine was degraded to form phosphocholine, glycerophosphocholine and free choline. No lysophosphatidylcholine accumulated. Sphingomyelin was degraded to form phosphocholine and a small amount of free choline. Magnesium ions stimulated choline production, while zinc ions were a potent inhibitor. Other divalent cations (calcium, manganese) had little effect on choline accumulation. ATP concentrations in brain homogenates were less than 5 nmol/mg protein (rapidly microwaved brain contained 27 nmol/mg protein). Addition of ATP or ADP to brain homogenates increased ATP concentrations and significantly inhibited choline accumulation. ATP diminished the formation of choline from added phosphatidylcholine, lysophosphatidylcholine, phosphocholine and glycerophosphocholine. The effects of ATP, zinc ion, or magnesium ion upon choline accumulation were not mediated by changes in the rates of utilization of choline for formation of phosphocholine or phosphatidylcholine. In summary, we showed that there was enhanced formation of choline when ATP concentrations within brain were low. This choline was derived, in part, from the degradation of phosphatidylcholine, and we suggest that phospholipase A activity was the primary initiator of choline release from this phospholipid.     

Effect of citrate on the different reactions catalyzed by rat mammary gland acetyl CoA carboxylase

The effect of citrate on the different reactions catalyzed by rat mammary gland acetyl CoA carboxylase has been investigated. Citrate showed modest effect on the ATP-orthophosphate and ATP-ADP exchange reactions. In contrast, this tricarboxylic acid caused marked concentration-dependent stimulation of the acetyl CoA-malonyl CoA exchange reaction which was concomitant with the activation of acetyl CoA carboxylation. The data obtained are consistent with the suggestion that activation by citrate of the overall forward reaction (malonyl CoA synthesis) primarily reflects enhancement of the carboxyltransferase half-reaction catalyzed by rat mammary gland acetyl CoA carboxylase.     

Inhibition of the synthesis of acetylcholine in rat brain slices by (-)-hydroxycitrate and citrate

Abstract: Slices of rat caudate nucleus were incubated in a solution of 123 mM-NaCl, 5 mM-KCl, 1.2 mM-MgCl₂, 1.2 mM-NaH₂PO₄, 25 mM-NaHCO₃, 0.2 mM-choline chloride, 0.058 mM-paraoxon, 1 mM-EGTA, and oxidizable substrates. (−)-Hydroxycitrate, a specific inhibitor of ATP-citrate lyase (EC 4.1.3.8), used at a concentration of 2.5 mM, inhibited the synthesis of acetylcholine (ACh) from [1,5-¹⁴C]citrate by 82–86%, but that from [U-¹⁴C]glucose by only 33%, from [2-¹⁴C]pyruvate by 24% and from [1-¹⁴C-acetyl]carnitine by 8%; the production of ¹⁴CO₂ from these substrates was not substantially changed. The synthesis of ACh from glucose and pyruvate was in hibited also by citrate; 2.5 mM- and 5 mM-citrate diminished it by 43% and 66%, respectively; the production of from [U-¹⁴C]glucose and from [1-¹⁴C]pyruvate was not affected. The mechanism of the inhibitory effect of citrate on the synthesis of ACh is not clear; the possibility is discussed that citrate alters the intracellular milieu in cholinergic neurons by chelating the intracellular Ca²⁺ and decreases the supply of mitochondrial acetyl-CoA to the cytosol. The results with (−)-hydroxycitrate indicate that the cleavage of citrate by ATP-citrate lyase is not responsible for the supply of more than about one-third of the acetyl-CoA which is used for the synthesis of ACh when glucose or pyruvate are the main oxidizable substrates. This proportion may be even smaller, since (−)-hydroxycitrate possibly affects the synthesis of ACh from glucose and pyruvate by a mechanism (unknown) similar to that of citrate, rather than by the inhibition of ATP-citrate lyase.