Antibodies to Cholinergic Neurons in Alzheimer''s Disease

Alzheimer's disease (AD) is associated with degenerative changes in nuclei of the basal forebrain which provide most of the cholinergic input to the cortex and hippocampus and with a reduction in presynaptic cholinergic parameters in these areas. Although the etiology and pathogenesis of AD are not known, several reports indicate the involvement of immunological mechanisms. In the present work we examined the existence of antibodies in sera of AD patients that bind specifically to cholinergic neurons. As antigens we employed the purely cholinergic electromotor neurons of the electric fish Torpedo which are chemically homogeneous and cross-react antigenically with human and other mammalian cholinergic neurons. Our findings show that immunoglobulins from sera of AD patients bind to a specific antigen (molecular mass 200 kilodaltons) in the cell bodies and axons of Torpedo electromotor neurons and that the levels of such antibodies are significantly higher in AD patients than in controls. The possible role of these antibodies in the cholinergic dysfunction in AD and their diagnostic potential are discussed.     

Transport of Muscarinic Cholinergic Marker Protein Activities in Regenerating Sciatic Nerve of Rat

The transport characteristics of choline acetyltransferase (ChAT; EC 2.3.1.6), acetylcholinesterase (AChE; EC 3.1.1.7), and the muscarinic acetylcholine receptors (mAChR) were studied in perineurally sutured, regenerating rat sciatic nerve. At different times after repair, the sciatic nerve was ligated for 24 h, and the activities of the cholinergic marker proteins, as well as the binding capacity, were measured proximally and distally from the ligature. The number of bidirectionally transported receptors increased linearly up to 5 months postoperatively (6.1-33.6% and 5.6-25.6% of the control level proximal and distal to the ligature, respectively). The quantity of anterogradely transported ChAT reached a plateau 3 months postoperatively (74.9% of the control level), whereas the retrogradely transported enzyme was then only 34.7% of the control value. The activity of AChE increased linearly during nerve regeneration, and exceeded the control level after 4 months (121.0% and 63.7% proximally and distally, respectively). The data indicate that the altered bidirectional transport of cholinergic marker proteins may be monitored quantitatively during nerve regeneration. This method might be suitable for studies of the nerve regeneration process.     

A Library of Monoclonal Antibodies to Torpedo Cholinergic Synaptosomes

Abstract: A library of monoclonal antibodies was generated to the cholinergic synaptosome. The immunogen was a preparation of highly purified synaptosomes from Torpedo electric organ. One hundred forty-one hybridoma cell lines were generated from the fusion of a single mouse. Tests reveal these cells produce antibodies with a vast range of neuronal specificities. The initial screen for specificity of antibody production was solid phase radioimmune binding to the original, highly purified synaptosome preparation. Subsequent tissue specificity tests have indicated that most antibodies are synaptosome-specific amongst the fish tissues tested: brain, liver, and purified synaptic vesicles. Less than 11% cross-react with liver. Many antibodies cross-react with frog and rat CNS. Localization within the frog and rat nervous tissue has revealed a vast array of antibody staining patterns. Some antibodies stain in a synaptic fashion. A few stain a restricted set of mammalian CNS neurons. Others define a broader set of CNS neurons. These Torpedo antibodies promise to be valuable probes with which to describe the molecular cell biology of the nervous system, of neurons in general, and of cholinergic neurons in particular.     

Identification of a Proteoglycan Antigen Characteristic of Cholinergic Synaptic Vesicles

An antiserum to cholinergic synaptic vesicles isolated from the electric organ of Torpedo marmorata was purified by adsorption with fractions containing unwanted antigens. The adsorbed antiserum responds to the proteoglycan core material of the cholinergic synaptic vesicles. The major antigen migrates in an anomalous fashion on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), forming a broad band with an apparent molecular weight of approximately 120,000 - 300,000. The distribution of this antigen after sucrose density gradient centrifugation of synaptic vesicles is the same as that of vesicular ATP. The antigen comigrates with a substance that can be stained with Alcian-Blue after SDS-PAGE of highly purified synaptic vesicles. This substance is related to the low-molecular-weight, Alcian-Blue-positive glycosaminoglycan vesiculin, which is formed from the high-molecular-weight proteoglycan by prolonged dialysis against water or by protease treatment. No antibodies were detected against vesiculin itself, indicating that the antigenic determinants are restricted to the proteoglycan.     

Acetyl-CoA Synthesizing Enzymes in Cholinergic Nerve Terminals

The activities of five enzymes involved in acetyl-CoA synthesis, pyruvate dehydrogenase complex, ATP citrate lyase, carnitine acetyltransferase, acetyl-CoA synthetase, and citrate synthase, were determined in normal nucleus interpeduncularis and nucleus interpeduncularis in which cholinergic terminals were removed following lesion of the habenulointerpeduncular tract. The activities of aspartate transaminase, fumarase, and GABA transaminase also were determined to compare the effect of lesion on other mitochondrial enzymes which are not linked to the biosynthesis of ACh. In normal nucleus interpeduncularis the activities of carnitine acetyltransferase and pyruvate dehydrogenase complex were higher than the activity of ChAT (choline acetyltransferase), whereas the activities of acetyl-CoA synthetase and citrate synthase were considerably lower than that of ChAT. The effect of the lesion separated the enzymes into two groups: the activities of pyruvate dehydrogenase complex, carnitine acetyltransferase, fumarase and aspartate transaminase decreased by 30--40%, whereas the activities of the other enzymes descreased 5--15%. ChAT activity was in all cases less than 15% of normal. It could be concluded that none of the acetyl-CoA synthesizing enzymes decreased to the degree that ChAT did. Only pyruvate dehydrogenase complex and carnitine acetyltransferase seem to be localized in cholinergic terminals to a significant degree. ATP citrate lyase as well as acetyl-CoA synthetase seem to have less significance in supporting acetyl-CoA formation in cholinergic nerve terminals.     

Endogenous ADP-Ribosylation in Brain: Initial Characterization of Substrate Proteins

Cholera and pertussis toxin-mediated ADP-ribosylation has been used extensively to study regulation of guanine nucleotide binding proteins (G proteins) in the nervous system, but much less is known about possible endogenous ADP-ribosylation of G proteins in brain. The present study demonstrates endogenous ADP-ribosylation, in the absence of cholera and pertussis toxins, of four predominate proteins in homogenates of rat cerebral cortex. These proteins showed apparent molecular masses of 20, 42, 45, and 50 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The 42- and 45-kDa proteins comigrated precisely with the major cholera toxin-labeled bands. Furthermore, the endogenous ADP-ribosylated and cholera toxin-ADP-ribosylated bands yielded identical 32P-labeled peptide fragments by one-dimensional peptide mapping, indicating that they are probably the same proteins, presumably the alpha-subunits of Gs. In contrast, peptide maps of the 50-kDa protein, which migrated close to a 48-kDa cholera toxin-labeled band, demonstrated that this protein is distinct from the toxin-labeled band and from Gs alpha. Levels of endogenous ADP-ribosylation activity showed regional heterogeneity in brain, with a nearly threefold variation observed among the brain regions examined. Chronic administration (7 days) of corticosterone significantly increased overall levels of endogenous ADP-ribosylation, indicating that components of this system may be under hormonal control in vivo. Attempts to identify neurotransmitters or second messenger systems that regulate endogenous ADP-ribosylation activity in brain have so far been unsuccessful with one exception.(ABSTRACT TRUNCATED AT 250 WORDS)     

Presence of Vasoactive Intestinal Polypeptide-Like Immunoreactivity in the Cholinergic Electromotor System of Torpedo marmorata

Vasoactive intestinal polypeptide (VIP)-like immunoreactivity was detected in the cholinergic electro-motor system of Torpedo marmorata using a combination of immunohistochemical assays, radioimmunoassay, and HPLC. The immunohistochemical assays revealed that the distribution of VIP-like immunoreactivity in the electric lobes, electromotor nerves, and electric organ is comparable to that of the stable cholinergic synaptic vesicle marker vesicle-specific proteoglycan. Ligation of the electromotor nerves caused a marked accumulation of VIP-like immunoreactivity in the lobes (180%) and the proximal portions of the electromotor nerves (130%) and a decrease in the electric organ (-50%), when measured by radioimmunoassay using synthetic VIP (porcine sequence) as the standard. VIP-like immunoreactivity in extracts of electric lobes electromotor nerves, and electric organ was eluted from a semipreparative reverse-phase HPLC column as a single peak with a retention time similar to that of porcine VIP. Rechromatography at higher resolution on an analytical column indicated diversity between the molecular forms of VIP-like immunoreactivity extracted from electric lobe and electric organ, suggesting the possibility of posttranslational processing.     

Polypeptide Hormones and Chromatin-Associated Proteins Act as Acceptors for Cholera Toxin-Catalyzed ADP-Ribosylation

Abstract: Cholera toxin catalyzed the ADP-ribosylation of the pituitary protein hormones thyrotropin (TSH), lutropin (LH), follitropin (FSH), human chorionic gonadotropin (hCG). and corticotropin (ACTH)_1–24, and ADP-ribosylation of the basic proteins histone subfraction H₁ and protamine. Casein and phosvitin, acidic nuclear proteins, did not act as acceptors for toxin-catalyzed ADP-ribosylation. The isolated TSH A and B subunits were tested for their ADP-ribose acceptor activity. The TSH A subunit showed fourfold greater ADP-ribose acceptor activity than the TSH B subunit. The ADP-ribose acceptor protein protamine was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis following incubation with cholera toxin under ADP-ribosylating conditions. [³H]ADP-ribose incorporated into protein from [³H]NAD migrated with the acceptor protein protamine. In the absence of added acceptor protein, the [³H]ADP-ribose incorporated into protein migrated with the A₁ fragment of cholera toxin. Cholera toxin A and B subunits were isolated and tested for their ability to catalyze the transfer of ADP-ribose to protamine. The cholera toxin A subunit showed 50-fold greater ADP-ribosyltransferase activity than the B subunit. Our data indicate that a variety of adenohypophyseal hormones and regulatory proteins act as acceptors for toxin-catalyzed ADP-ribosylation. These studies may help in understanding the role of endogenous ADP-ribosyltransferases and the physiological effects of this modification of protein.     

Hydrodynamic Molecular Weight of Solubilized Cholinergic Synaptic Vesicle Glycoprotein ATPase

The Torpedo californica electric organ synaptic vesicle glycoprotein ATPase was solubilized with octaethyleneglycoldodecyl ether and stabilized with phosphatidylserine. The complex was analyzed by size exclusion chromatography and band sedimentation velocity ultracentrifugation in water/glycerol and deuterium oxide/glycerol density gradients. The complex was found to have a Stokes' radius of 79 +/- 0.7 A, a sedimentation velocity coefficient at 20 degrees C in water of 6.8 +/- 0.2S, a partial specific volume of 0.81 +/- 0.01 cm3/g, and a frictional coefficient of 1.6. The molecular weight of the solubilized complex was calculated to be 320,000 +/- 7,000 and that of the protein 210,000 +/- 9,000. The relationship of this latter value to the major transport ATPase types is discussed.     

Separation of Recycling and Reserve Synaptic Vesicles from Cholinergic Nerve Terminals of the Myenteric Plexus of Guinea Pig Ileum

Acetylcholine-rich synaptic vesicles were isolated from myenteric plexus-longitudinal muscle strips derived from the guinea pig ileum by the method of Dowe, Kilbinger, and Whittaker [J. Neurochem. 35, 993-1003 (1980)] using either unstimulated preparations or preparations field-stimulated at 1 Hz for 10 min using pulses of 1 ms duration and 10 V . cm-1 intensity. The organ bath contained either tetradeuterated (d4) choline (50 microM) or [3H]acetate (2 muCi . ml-1); d4 acetylcholine was measured by gas chromatography-mass spectrometry. As with Torpedo electromotor cholinergic vesicle preparations made under similar conditions the distribution of newly synthesized (d4 or [3H]) acetylcholine in the zonal gradient from stimulated preparations was not identical with that of endogenous (d0, [1H]) acetylcholine, but corresponded to a subpopulation of denser vesicles (equivalent to the VP2 fraction from Torpedo) that had preferentially taken up newly synthesized transmitter. The density difference between the reserve (VP1) and recycling (VP2) vesicles was less than that observed in Torpedo but this smaller difference can be accounted for theoretically by the difference in size between the vesicles of the two tissues. At rest, a lesser incorporation of labelled acetylcholine into the vesicle fraction was observed, and the peaks of endogenous and newly synthesized acetylcholine coincided. Stimulation in the absence of label followed by addition of label did not lead to incorporation of labelled acetylcholine, suggesting that the synthesis and storage of acetylcholine in this preparation and its recovery from stimulation is much more rapid than in Torpedo.     

Ecto-5''-Nucleotidase Is Associated with Cholinergic Nerve Terminals in the Hippocampus but Not in the Cerebral Cortex of the Rat

The extracellular catabolism of exogenously added AMP was studied in immunopurified cholinergic nerve terminals and in slices of the hippocampus and cerebral cortex of the rat. AMP (10 microM) was catabolized into adenosine and inosine in hippocampal cholinergic nerve terminals and in hippocampal slices, as well as in cortical slices. IMP formation from extracellular AMP was not detected. alpha, beta-Methylene ADP (100 microM) inhibited almost completely the extracellular catabolism of AMP in these preparations. The relative rate of catabolism of AMP was greater in hippocampal slices than in cortical slices. AMP was virtually not catabolized when added to immunopurified cortical cholinergic nerve terminals, although ATP could be catabolized extracellularly under identical conditions. The comparison of the relative rates of catabolism of exogenously added AMP, calculated from the amount of AMP catabolized after 5 min, in hippocampal cholinergic nerve terminals and in hippocampal slices revealed a nearly 50-fold enrichment in the specific activity of ecto-5'-nucleotidase upon immunopurification of the cholinergic nerve terminals from the hippocampus. The results suggest that there is a regional variation in the subcellular distribution of ecto-5'-nucleotidase activity in the rat brain, the ecto-5'-nucleotidase in the hippocampus being closely associated with the cholinergic nerve terminals, whereas in the cerebral cortex ecto-5'-nucleotidase activity seems to be located preferentially outside the cholinergic nerve terminals.     

Mg-ATPase and Torpedo Cholinergic Synaptic Vesicles

The reported presence of Mg-ATPase activity in cholinergic synaptic vesicles from the electric organ of Torpedo marmorata was reinvestigated in view of possible contamination of vesicles by other subcellular fractions. After dilution in concentrated sucrose, the vesicular fraction isolated on a sedimentation sucrose gradient was purified further on a flotation density gradient. It appears that this treatment allows separation of the vesicles according to their content. The two vesicular content markers, acetylcholine and ATP, are recovered as sharp coincident peaks at a density close to 0.48 M sucrose. Empty vesicles are identified in denser regions by the protein pattern on gel electrophoresis which is identical to the pattern obtained for filled vesicles. Refractionation of vesicles depleted of their acetylcholine content by valinomycin leads to an extreme picture, with a massive shift of the vesicles toward denser regions. We have then shown that a ouabain-insensitive Mg-ATPase is indeed associated with the vesicle membrane, but the activity is fully apparent only when vesicles are permeabilized either as the result of the fractionation procedure or after detergent treatment. The relative insensitivity of the Mg-ATPase associated with the synaptic vesicles to oligomycin, N,N'-dicyclohexylcarbodiimide, and azide indicates that this enzyme differs from the classic F1F0 mitochondrial enzyme. The most striking finding is the sensitivity to vanadate of the vesicular Mg-ATPase, which suggests the involvement of a phosphorylated intermediate. On the basis of both the difference in inhibitor sensitivity between untreated and detergent-treated vesicles and of the pronase experiments, the possibility that the enzyme has an inward orientation is discussed.     

Presence of a Membrane-Bound Acetylcholinesterase Form in a Preparation of Nerve Endings from Torpedo marmorata Electric Organ

Abstract: We adapted a method, originally described by Israel et al. (1976) for the preparation of cholinergic nerve endings from Torpedo , to deal with a larger quantity of electric tissue. We followed the distribution of acetylcholine (ACh), ATP, acetylcholine receptor (AChR), choline acetyltransferase (ChAT), ouabainresistant and -sensitive ATPase, lactate dehydrogenase (LDH) and acetylcholinesterase (AChE) and obtained a nerve ending fraction, without detectable contamination by postsynaptic components. This preparation consisted of closed structures of 1–5 μm diameter, containing synaptic vesicles. It had the capacity to synthetize and release ACh. This preparation is therefore quite suitable for biochemical analysis of presynaptic elements. We particularly investigated its content of AChE: it consists exclusively of the 6S dimeric, hydrophobic form of the enzyme. This enzyme is enriched in the nerve ending preparation, by a factor higher than that obtained for ChAT. The yields obtained for the two enzymes suggest that the hydrophobic 6S AChE form may be mostly presynaptic in Torpedo electric organs. We characterized this form as a membrane-bound, externally active enzyme in the nerve ending preparation. It may thus participate in the hydrolysis of extracellularly liberated AChE and its abundance suggests that presynaptic AChE could play an essential role in cholinergic transmission in Torpedo electric organs and perhaps also in other cholinergic synapses.     

Synapsin I Is Associated with Cholinergic Nerve Terminals in the Electric Organs of Torpedo, Electrophorus, and Malapterurus and Copurifies with Torpedo Synaptic Vesicles

Using an affinity-purified monospecific polyclonal antibody against bovine brain synapsin I, the distribution of antigenically related proteins was investigated in the electric organs of the three strongly electric fish Torpedo marmorata, Electrophorus electricus, Malapterurus electricus and in the rat diaphragm. On application of indirect fluorescein isothiocyanate-immunofluorescence and using alpha-bungarotoxin for identification of synaptic sites, intense and very selective staining of nerve terminals was found in all of these tissues. Immunotransfer blots of tissue homogenates revealed specific bands whose molecular weights are similar to those of synapsin Ia and synapsin Ib. Moreover, synapsin I-like proteins are still attached to the synaptic vesicles that were isolated in isotonic glycine solution from Torpedo electric organ by density gradient centrifugation and chromatography on Sephacryl-1000. Our results suggest that synapsin I-like proteins are also associated with cholinergic synaptic vesicles of electric organs and that the electric organ may be an ideal source for studying further the functional and molecular properties of synapsin.     

Purification and Subunit Composition of a Cholinergic Synaptic Vesicle Glycoprotein, Phosphointermediate-Forming ATPase

A glycoprotein ATPase in cholinergic synaptic vesicles of Torpedo electric organ was solubilized with octa-ethylene glycol dodecyl ether detergent. Study of potential stabilizing factors identified crude brain phosphatidylserine, glycerol, dithiothreitol, and protease inhibitors as of value in maintaining activity. The ATPase was purified from the solubilized, stabilized material by glycerol density gradient band sedimentation velocity ultracentrifugation, and hydroxylapatite, wheat germ lectin affinity, and size exclusion chromatographies. The pure ATPase had a specific activity of about 37 mumol ATP hydrolyzed/min/mg protein. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the purified material typically exhibited three polypeptides of molecular masses 110, 104, and 98 kilodaltons (kDa) and a fourth diffuse polypeptide of 60 kDa. This composition suggests that the ATPase is a member of the P-type, or phosphointermediate-forming, family, but it was shown to be distinct from the ouabain-sensitive Na+,K+- and CA2+-stimulated Mg2+-ATPases. The purified vesicle enzyme was rapidly phosphorylated by [gamma-32P]ATP on about 14% of the subunits with molecular weights of 98,000-110,000. About 16% of the ATPase was phosphorylated in whole-vesicle ghosts in a manner consistent with formation of a phosphointermediate, thus confirming the P-type nature of this enzyme.     

The Structural Specificity of Choline Transport into Cholinergic Nerve Terminals

Abstract: The accumulation of choline, homocholine, and 4-hydroxybutyl-trimethylammonium by rat brain synaptosomes was measured; the choline uptake mechanism transported homocholine but not hydroxybutyltrimethylammonium, which, in addition, did not block choline accumulation. In cats'superior cervical ganglia, preganglionic nerve stimulation increased the accumulation of homocholine, but not that of hydroxybutyltrimethylammonium. It is concluded that the substrate specificity of the choline transport mechanism is such that increasing the N-O atom distance by one methylene group retains affinity, but increasing this distance by two methylene groups does not.     

Identification of GTP-Binding Proteins by ADP-Ribosylation in the Presence of Cholera Toxin, Pertussis Toxin and Botulinum Toxin D in Plasma Membrane Isolated from Eggs and Embryos of Sea Urchin

In plasma membrane fraction isolated from eggs and embryos of sea urchin, ³²P-labeled proteins were found on the fluorographs of SDS-polyacrylamide gel electrophoresis, performed after an exposure of the fraction to [adenylate-³²P] nicotinamide adenine dinucleotide in the presence of cholera toxin, pertussis toxin or botulinum toxin D. The molecular weights of proteins, thus ADP-ribosylated in the presence of cholera toxin and pertussis toxin are 45 and 39 K, which correspond to Gs and Gi or Go, respectively. Protein with the molecular weight of 24 K, labeled in the presence of botulinum toxin D, corresponds to small molecular weight G-protein. The labeling intensity of 45 K protein, probably proportional to its amount, became high at the blastula stage. The labeling intensity of 39 K protein was hardly altered up to the blastula stage. The labeling intensity of 24 K protein increased after fertilization and further increase occurred at the blastula stage. At the gastrula stage, the labeling intensities of these proteins became somewhat lower than at the blastula stage. Transmembrane signaling system, in which these G-proteins are involved, is probably altered in its function during early development.     

Effect of Aluminum on Acetyl-CoA and Acetylcholine Metabolism in Nerve Terminals

Abstract: The potential ability of Al to affect cholinergic transmission was studied on synaptosomal fractions of rat brain incubated with pyruvate in depolarizing medium containing 30 m M K⁺. Addition of 1 m M Ca caused a 266% increase in the acetylcholine (ACh) release despite decreased pyruvate oxidation. Under these conditions, 0.25 m M Al did not affect pyruvate oxidation but raised mitochondrial and decreased synaptoplasmic acetyl-CoA. Simultaneously, a 61% inhibition of Ca-evoked ACh release was observed. Verapamil (0.1 and 0.5 m M ) decreased the acetyl-CoA concentration in synaptoplasm and inhibited ACh release. Al (0.012 m M ) partially reversed these inhibitory effects. Omission of P_i from the medium abolished suppressive effects of Al on acetyl-CoA content and Ca-evoked transmitter release. We conclude that the Al(PO₄)OH⁻ complex may be the active form of Al, which, by interaction with the verapamil binding sites of Ca channels, is likely to restrict the Ca influx to the synaptoplasm. This may inhibit the provision of acetyl-CoA to the synaptoplasm as well as the Ca-evoked ACh release. One may suppose that excessive accumulation of Al in some encephalopathic brains may, by this mechanism, suppress still-surviving cholinergic neurons and exacerbate cognitive deficits caused by already-existing structural losses in the cholinergic system.     

Ectonucleotidase Activities Associated with Cholinergic Synaptosomes Isolated from Torpedo Electric Organ

Intact synaptosomes isolated from the electric organ of the electric ray Torpedo marmorata contain, at their surface, enzyme activities for the hydrolysis of externally applied nucleoside phosphates. The diazonium salt of sulfanilic acid, as a low-molecular-weight, slowly permeating, covalent inhibitory agent, selectively blocks these enzyme activities and leaves intracellular lactate dehydrogenase intact. The ectoenzymes comprise both a nucleoside triphosphate and diphosphate phosphohydrolase, as well as a 5'-nucleotidase. Activity of nonspecific ectophosphatases is absent. The nucleoside triphosphatase hydrolyzes almost equally well ATP, GTP, CTP, UTP, and ITP and is activated to a similar degree by Mg2+ or Ca2+. It has a high affinity for ATP (Km for ATP in the presence of Mg2+, 75 microM; in the presence of Ca2+, 66 microM). Maximal rates in the presence of Mg2+ and Ca2+ were very similar (34.8 and 32.5 nmol of Pi/min/mg of synaptosomal protein, respectively). Either Mg-ATP or Ca-ATP can act as a true substrate. ADP inhibits hydrolysis of ATP, but AMP is without effect. The nucleoside triphosphatase is not inhibited significantly by a number of inhibitors of mitochondrial Mg2+-ATPase or of Ca2+ + Mg2+-ATPases. It is, however, considerably inhibited by filipin and quercitin. The capacity of intact synaptosomes to hydrolyze also extracellular ADP, GDP, AMP, GMP, and IMP suggests that the nucleoside triphosphatase is part of an enzyme chain that causes complete hydrolysis of the respective nucleoside triphosphate to the nucleoside. We conclude that the cholinergic nerve terminals of the Torpedo electric organ can hydrolyze ATP released on coexocytosis with acetylcholine via an ectonucleoside triphosphatase activity that is different from known endogenous nerve terminal ATPases. The final product of the hydrolysis, adenosine, can then be salvaged by the nerve terminal for resynthesis of ATP. Other possible physiological functions of the ectonucleotidases are discussed.     

Differential Effects of Nerve Growth Factor on Expression of Choline Acetyltransferase and Sodium-Coupled Choline Transport in Basal Forebrain Cholinergic Neurons in Culture

Abstract: Nerve growth factor (NGF) treatment of primary cultures of embryonic day 17 rat basal forebrain differentially altered activity of choline acetyltransferase (ChAT) and high-affinity choline transport; ChAT specific activity was increased by threefold in neurons grown in the presence of NGF for between 4 and 8 days, whereas high-affinity choline transport activity was not changed relative to control. Dose-response studies revealed that enhancement of neuronal ChAT activity occurred at low concentrations of NGF with an EC₅₀ of 7 ng/ml, with no enhancement of high-affinity choline transport observed at NGF concentrations up to 100 ng/ml. In addition, synthesis of acetylcholine (ACh) and ACh content in neurons grown in the presence of NGF for up to 6 days was increased significantly compared with controls. These results suggest that regulation of ACh synthesis in primary cultures of basal forebrain neurons is not limited by provision of choline by the high-affinity choline transport system and that increased ChAT activity in the presence of NGF without a concomitant increase in high-affinity choline transport is sufficient to increase ACh synthesis. This further suggests that intracellular pools of choline, which do not normally serve as substrate for ACh synthesis, may be made available for ACh synthesis in the presence of NGF.