Phorbol Esters Induce Neurotransmitter Release in Cholinergic Synaptosomes from Torpedo Electric Organ

The effect of phorbol esters and so the involvement of Ca2+/phospholipid-dependent protein kinase (protein kinase C;PKC) in the release of acetylcholine (ACh) was studied using Torpedo electric organ synaptosomes. 12-O-Tetradecanoylphorbol 13-acetate (TPA), a known activator of PKC, induced neurotransmitter release in a concentration-dependent manner and increased the potassium-evoked release of ACh. The effect of TPA was shown to be independent of the extrasynaptosomal calcium concentration. TPA-induced ACh release was reversed by H-7, an inhibitor of PKC activity. This drug showed no effect on potassium-evoked ACh release. Botulinum toxin, a strong blocker of potassium-induced ACh release in that synaptosomal preparation, showed no inhibitory effect on the TPA-induced ACh release. Our results suggest that activation of PKC potentiates the release of an ACh pool that is not releasable by potassium depolarization, independently of the extracellular calcium concentration.     

Continuous Determination by a Chemiluminescent Method of Acetylcholine Release and Compartmentation in Torpedo Electric Organ Synaptosomes

Abstract: The detection of acetylcholine (ACh) with a chemiluminescent procedure enables one to follow continuously the release of transmitter from stimulated synaptosomes and to study the compartmentation of ACh in resting and active nerve terminals. A compartment of ACh liberated almost entirely by a single freezing and thawing could be directly measured and compared with a compartment of ACh resistant to several cycles of freezing and thawing but liberated by a detergent (60–70% of the total). It is the compartment liberated by freezing and thawing that is reduced when synaptosomes are stimulated. Up to half the total synaptosomal ACh content is readily releasable provided the calcium entry is maintained, or if a strong releasing agent such as the venom of Glycera convoluta is used. In addition, it is shown that synaptosomes contain only negligible amounts of choline, and that the proportion of the two ACh compartments is not influenced by changing extracellular calcium just before their determination.     

Calcium-Independent Release of Acetylcholine from Electric Organ Synaptosomes and Its Changes by Depolarization and Cholinergic Drugs

Chemiluminescent detection was applied to measure the continuous spontaneous Ca2+-independent liberation of acetylcholine (ACh) from Torpedo electric organ synaptosomes. Differentiation between the release of ACh and choline was achieved by inhibiting cholinesterases with phospholine, and a way to quantify the continuous release was devised. The method permitted measurements during short time intervals from minute amounts of tissue and without an accumulation of ACh in the medium. Synaptosomes continuously liberated small amounts of ACh during incubations in the presence of 3 mM K+ and in the absence of Ca2+. The spontaneous liberation of ACh was similar both quantitatively and qualitatively at pH values of 8.6 and 7.8. It was unaltered by MgCl2 (10.4 mM), 2-(4-phenylpiperidino)cyclohexanol (10 microM), ouabain (104 microM), atropine (10 microM), and valinomycin (102 nM). Carbamoylcholine brought about a decrease, which could be partially reversed by atropine. The Ca2+-independent output of ACh was increased considerably when the concentration of K+ ions was raised (eightfold at 103 and 35-fold at 203 mM K+). Carbamoylcholine (104 microM) blocked the increase in ACh release produced by high K+; this effect of carbamoylcholine was not reversed by atropine (10 microM). When Ca2+ was added to synaptosomes depolarized by a high concentration of K+, the amount of ACh released during the first 1-3 min after the addition of Ca2+ was at least 20 times higher than in the absence of Ca2+, but the release returned rapidly to predepolarization values. Similarly high values of ACh release could be achieved by adding Ca2+ plus the ionophore A23187 and even higher values by adding Ca2+ plus gramicidin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thiamine and Cholinergic Transmission in the Electric Organ of Torpedo

The electric organ of Torpedo marmorata was found to contain as much as 120 +/- 24 nmol of thiamine per g of fresh tissue. The vitamin was distributed as nonesterified thiamine (32%), thiamine monophosphate (22%), thiamine diphosphate (8%), and an important proportion of thiamine triphosphate (38%). A high level of thiamine triphosphate was found in synaptosomes isolated from the electric organ. In contrast, the synaptic vesicles did not show any enrichment in thiamine, whereas they contained a marked peak of acetylcholine (ACh) and ATP. Thus thiamine seems to be very abundant in cholinergic nerve terminals; its localization is apparently extravesicular, either in the axoplasm or in association with plasma membrane. When calcium was reduced and magnesium increased in the external medium, the efficiency of transmission was diminished, owing to inhibition of ACh release; in a parallel manner the degree of thiamine phosphorylation was found to increase--this condition is known to modify the repartition of ACh between vesicular and extravesicular compartments. Electrical stimulation, which causes periodic variations of the level of ACh and ATP, also caused significant changes in thiamine esters. In addition, related changes of the vitamin and the transmitter were observed under other conditions, suggesting a functional link between the metabolism of thiamine and that of ACh in cholinergic nerve terminals.     

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.     

Newly Synthesized and Preformed Acetylcholine Are Released from Torpedo Synaptosomes by Different Pathways

In this study, we investigated the mechanisms underlying the release of preformed and of newly synthesized acetylcholine (ACh) from isolated Torpedo nerve terminals (synaptosomes). This was pursued by examining and comparing the effects of anticytoskeletal and anticalmodulin drugs and of activating the presynaptic muscarinic ACh receptors on the release of preformed endogenous ACh and of newly synthesized radiolabeled ACh. The anticytoskeletal drugs vinblastine, cytochalasin B, and colchicine inhibit the Ca2+-dependent K+-mediated release of newly synthesized radiolabeled ACh, but have no effect on the release of preformed ACh. By contrast, the muscarinic agonist oxotremorine markedly inhibits the release of preformed ACh, but has little effect on the release of newly formed ACh. Treatment of the synaptosomes with the calmodulin antagonist trifluoperazine inhibits the release of both ACh pools concomitantly. These findings show that preformed and newly synthesized ACh are released by different routes and suggest that their secretion is mediated by converging pathways. The significance of these results in view of the previously demonstrated preferential release of newly synthesized ACh is discussed.     

Induction of 5-Deiodinase Activity in Astroglial Cells by 12-O-Tetradecanoylphorbol 13-Acetate and Fibroblast Growth Factors

In the brain, 5'-deiodinase (5'-D) is responsible for the metabolic activation of thyroxine (T4) into 3,5,3'-triiodothyronine (T3) and 5-deiodinase (5-D) deiodinates T4 and T3 into inactive metabolites. This study examines the effects of factors known to induce astroglial 5'-D activity on the 5-D activity in cultured rat astroglial cells. The potencies of these factors were compared after 8 h of incubation, when stimulations by these factors near their maximal effects. 12-O-Tetradecanoylphorbol 13-acetate (TPA) at 10(-7) M was a potent inducer of 5-D activity, producing a 30- to 80-fold increase after 8 h. The maximal effect of TPA was observed after about 14 h. The TPA stimulation of 5-D activity was not dependent on glucocorticoids, unlike 5'-D activity. In comparison with TPA, 8-bromo-cyclic AMP (10(-3) M) was a poor inducer of 5-D activity whereas it is an excellent inducer of 5'-D activity. It produced a 2- to 20-fold increase in 5-D activity after 8 h. Natural acidic fibroblast growth factor (20 ng/ml) produced a degree of stimulation similar to that of TPA after 8 h. The maximal effect of acidic fibroblast growth factor was observed after about 16 h (until a 120-fold increase). Recombinant acidic fibroblast growth factor also induced 5-D activity. Basic fibroblast growth factor was less potent than acidic fibroblast growth factor for increasing 5-D activity (maximal increase by 40- to 50-fold after 8 h).(ABSTRACT TRUNCATED AT 250 WORDS)     

Compared Effects of Two Vesicular Acetylcholine Uptake Blockers, AH5183 and Cetiedil, on Cholinergic Functions in Torpedo Synaptosomes: Acetylcholine Synthesis, Choline Transport, Vesicular Uptake, and Evoked Acetylcholine Release

We examined the effects of two drugs, AH5183 and cetiedil, demonstrated to be potent inhibitors of acetylcholine (ACh) transport by isolated synaptic vesicles on cholinergic functions in Torpedo synaptosomes. AH5183 exhibited a high specificity toward vesicular ACh transport, whereas cetiedil was shown to inhibit both high-affinity choline uptake and vesicular ACh transport. Choline acetyltransferase was not affected by either drug. High external choline concentrations permitted us to overcome cetiedil inhibition of high-affinity choline transport, and thus synthesis of [14C]ACh in treated preparations was similar to that in controls. We then tested evoked ACh release in drug-treated synaptosomes under conditions where ACh translocation into the vesicles was blocked. We observed that ACh release was impaired only in cetiedil-treated preparations; synaptosomes treated with AH5183 behaved like the controls. Thus, this comparative study on isolated nerve endings allowed us to dissociate two steps in drug action: upstream, where both AH5183 and cetiedil are efficient blockers of the vesicular ACh translocation, and downstream, where only cetiedil is able to block the ACh release process.     

Origin of the Bicarbonate Stimulation of Torpedo Electric Organ Synaptic Vesicle ATPase

Abstract: The origin of the stimulation by bicarbonate of the ATPase found in cholinergic synaptic vesicles isolated from Torpedo californica electric organ was studied. Given in descending order of potency, the anions formate, malonate, acetate, thiosulfate, cyanate, biphosphite, biselenite, and bisulfite also stimulate to lesser extents. The anion channel blockers 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS) and 4,4'-diisothiocyanostil-bene-2,2'-disulfonic acid (DIDS) inactivated the ATPase at micromolar concentrations, but there was no indication that the reagents acted to block an anion channel. The LineweaverBurk plot of the basal ATPase activity was biphasic, indicating high- and low–affinity states with K_ms of 0.0098 ' 0.0013 and 1.2 ' 0.1 mM for V _maxs of 52 ' 2 and 82 ' 9 nmol/min/mg protein, respectively. It became nearly linear in the presence of bicarbonate, indicating conversion of all of the ATPase to one high affinity-high velocity state with a K _m of 0.054 ' 0.009 mM and a V _max of 232 ' 0.051 nmol/min/mg protein. The pH activity profile for basal ATPase was bell-shaped and optimal between pH 6 and 8. The bicarbonate stimulation was maximal at about pH 7. The ATPase could be solubilized in 1.6% Lubrol WX with full retention of the bicarbonate stimulation. Although ouabain and oligomycin act at high concentrations to inhibit bicarbonate stimulation of the ATPase, there is no contamination by the Na⁺-K⁺ ATPase, mitochondrial ATPase, or other common ATPases. The study suggests that bicarbonate acts to stimulate the ATPase by acting directly on it at an allosteric site on the outside of the synaptic vesicle.     

Neurite Outgrowth and Protein Phosphorylation in Chick Embryonic Sensory Ganglia Induced by a Brief Exposure to 12-O-Tetradecanoylphorbol 13-Acetate

Abstract: An exposure to 12- O -tetradecanoylphorbol 13-acetate (TPA) at 20 n M for as short as 30 min was sufficient to elicit neurite outgrowth from explanted chick embryonic sensory ganglia. Attachment of the ganglia to the collagencoated substratum during exposure to TPA was essential for subsequent neurite outgrowth. Pulse-labeling with [³⁵S]-methionine indicated no significant difference in protein synthesis between control and TPA-treated ganglia. In vitro phosphorylation assay revealed a prominent protein kinase C substrate with an apparent molecular mass of 66,000 dalton (66 kDa) in chick embryo ganglia extracts. Treatment of intact ganglia with TPA for 30 min also specifically stimulated the phosphorylation of the same protein. When staurosporine, a potent inhibitor of protein kinase C, was present during TPA treatment, both neurite outgrowth and the phosphorylation of the 66-kDa protein were blocked. Biochemical analysis of the phosphorylated 66-kDa protein indicated that (1) phosphorylation was only in serine residue, (2) the pI value was 4.5, (3) after V8 protease digestion, two phosphorylated peptide fragments, 6.0 and 7.5 kDa in size, were produced, and (4) it cross-reacted with an antiserum raised against a 66-kDa neurofilament subunit from rat spinal cord. These results suggest that early activation of protein kinase C and the phosphorylation of the 66-kDa protein may be involved in neuritogenesis.     

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.     

Energy Metabolism and Quantal Acetylcholine Release: Effects of Botulinum Toxin, l-Fluoro-2,4-Dinitrobenzene, and Diamide in the Torpedo Electric Organ

In the Torpedo electric organ, a modified nerve-muscle system, type A botulinum toxin blocked the release of acetylcholine (ACh) quanta, both neurally evoked and spontaneous. At the same time, the toxin increased the release of a class of small miniature potentials (the subminiature potentials), reduced the ATP and more the creatine phosphate content of the tissue, and impaired the activity of creatine kinase (CK). Thus, we compared this pattern of changes with those provoked by 1-fluoro-2,4-dinitrobenzene (FDNB), an efficient inhibitor of CK. As expected, FDNB rapidly inactivated CK, which resulted in a profound depletion of ATP whereas the stores of creatine phosphate were preserved. In addition, FDNB caused conspicuous morphological alterations of nerve endings and ACh depletion. This agent also suppressed evoked and spontaneous quantal release whereas the occurrence of subminature potentials was markedly increased. Diamide, a penetrating thiol oxidizing substance, provoked first a transient rise in quantal ACh release and then blockade of transmission with, again, production of a large number of subminiature potentials. Creatine phosphate was depleted in the tissue by diamide, the ATP content reduced, and CK activity partly inhibited. The morphology of nerve terminals did not show obvious changes with either diamide or botulinum toxin at the stage of transmission failure. Although the three poisons acted by different mechanisms, this resulted in a rather similar pattern of physiological changes: failure of quantal release and enhancement of subquantal release. These results and experiments on synaptosomes indicated that CK inhibition was probably a crucial mechanism for FDNB but not for diamide or botulinum intoxication.(ABSTRACT TRUNCATED AT 250 WORDS)     

The Synthesis, Storage, and Release of Propionylcholine by the Electric Organ of Torpedo marmorata

Abstract: Little is known about the specificity of the mechanisms involved in the synthesis and release of acetylcholine for the acetyl moiety. To test this, blocks of tissue from the electric organ of Torpedo were incubated with either [1-¹⁴C]acetate or [1-¹⁴C]propionate, and the synthesis, storage, and release of [1-¹⁴C]acetylcholine and [¹⁴C]propionylcholine were compared. To obtain equivalent amounts of the two labeled choline esters, a 50-fold higher concentration of propionate than of acetate was needed. Following subcellular fractionation, similar proportions of [¹⁴C]acetylcholine and [¹⁴C]propionylcholine were recovered with synaptosomes and with synaptic vesicles. Furthermore, both labeled choline esters were protected to a similar extent from degradation during homogenization of tissue in physiological medium, indicating that the two choline esters were equally well incorporated into synaptic vesicles. Yet depolarization of tissue blocks by 50 m M KCI released much less [¹⁴C]propionylcholinc than [¹⁴C]acetylcholine. During field stimulation of the tissue blocks, the difference between the releasibility of the two choline esters was less marked, but acetylcholine was still released in preference to propionylcholine. Evidence for specificity of the release mechanism was also obtained when the release of the two choline esters in response to field stimulation was compared in tissue blocks preincubated with both [³H]choline and [¹⁴C]propionate.     

Uptake of Acetate and Propionate by Isolated Nerve Endings from the Electric Organ of Torpedo marmorata and Their Incorporation into Choline Esters

Abstract: The uptake and incorporation into choline esters of acetate and propionate by electric organ synaptosomes were compared, with the aim of better understanding the basis for the selectivity of choline ester synthesis shown by this tissue for acetate. It was found that propionate uptake, like acetate uptake, was a temperature-dependent, saturable process. Both uptake mechanisms had similar affinities for their substrates, but the maximal velocity of propionate uptake was considerably lower than that of acetate uptake; and less of the accumulated propionate was used for choline ester synthesis than of the accumulated acetate. While acetate was a good inhibitor of propionate uptake, propionate was a very poor inhibitor of acetate uptake. This finding, in addition to the observation that the two uptakes were not affected in the same way by changes in pH, led to the suggestion that acetate uptake and propionate uptake reflect different processes. In both cases, however, the pH dependence of uptake indicated that these substrates cross the membrane as the charged species. Acetate uptake and acetylcholine synthesis remained closely associated under various experimental conditions, while propionate uptake could be dissociated from the synthesis of propionylcholine. Hence, it appears that acetate is taken up by a specific, high-velocity mechanism linked to acetylcholine synthesis, whereas propionate uptake may represent a less specific mechanism.     

Inhibition by Cyclic AMP of Phorbol Ester-Potentiated Norepinephrine Release from Guinea Pig Brain Cortical Synaptosomes

The involvement of Ca2+/phospholipid-dependent protein kinase (protein kinase C, PKC) and cyclic AMP-dependent protein kinase in the K+-evoked release of norepinephrine (NE) was studied using guinea pig brain cortical synaptosomes preloaded with [3H]NE. 12-O-Tetradecanoylphorbol-13-acetate (TPA), a potent activator of PKC, enhanced the K+-evoked release of [3H]NE, in a concentration-dependent manner, but with no effect on the spontaneous outflow and uptake of [3H]NE in the synaptosomes. The apparent affinity of the evoked release for added calcium but not the maximally evoked release was increased by TPA (10(-7) M). Inhibitors of PKC, polymyxin B, and a more potent inhibitor, staurosporine, counteracted the TPA-induced potentiation of the evoked release. Both forskolin and dibutyryl cyclic AMP (DBcAMP) enhanced the evoked release, but reduced the TPA-potentiated NE release. A novel inhibitor of cyclic AMP-dependent protein kinase, KT5720, blocked both the forskolin-induced increase in the evoked release and its inhibition of TPA-induced potentiation in the evoked release, thereby suggesting that forskolin or DBcAMP counteracts the Ca2+-dependent release of NE by activating cyclic AMP-dependent protein kinase. These results suggest that the activation of PKC potentiates the evoked release of NE and that the activation of cyclic AMP-dependent protein kinase acts negatively on the PKC-activated exocytotic neurotransmitter release process in brain synaptosomes of the guinea pig.     

Voltage-Dependent Anion Channel Proteins in Synaptosomes of the Torpedo Electric Organ: Immunolocalization, Purification, and Characterization

In this study, we purified and characterized the voltage-dependent anion channel (VDAC) from the Torpedo electric organ. Using immunogold labeling, VDAC was colocalized with the voltage-gated Ca²⁺ channel in the synaptic plasma membrane. By immunoblot analysis, five protein bands in synaptosomes isolated from the Torpedo electric organ cross reacted with two monoclonal anti-VDAC antibody. No more than about 7 to 10% mitochondrial contains could be detected in any synaptosomal membrane preparation tested. This was estimated by comparing the specific activity in mitochondria and synaptosomes of succinate–cytochrome-c oxidoreductase and antimycin-insensitive NADH–cytochrome-c oxidoreductase activities; mitochondrial inner and outer membrane marker enzymes, respectively. [¹⁴C]DCCD (dicyclohexylcarbodiimide), which specifically label mitochondrial VDAC, labeled four 30–35 kDa protein bands that were found to interact with the anti-VDAC antibody. The distribution of the Torpedo VDAC protein bands was different among membranes isolated from various tissues. VDAC was purified from synaptosomes and a separation between two of the proteins was obtained. The two purified proteins were characterized by their single channel activity and partial amino acid sequences. Upon reconstitution into a planar lipid bilayer, the purified VDACs showed voltage-dependent channel activity with properties similar to those of purified mitochondrial VDAC. Amino acid sequence of four peptides, derived from VDAC band II, exhibited high homology to sequences present in human VDAC1 (98%), VDAC2 (91.8%), and VDAC3 (90%), while another peptide, derived from VDAC band III, showed lower homology to either VDAC1 (88.4%) or VDAC2 (79%). Two more peptides show high homology to the sequence present in mouse brain VDAC3 (100 and 78%). In addition, we demonstrate the translocation of ATP into synaptosomes, which is inhibited by DCCD and by the anion transport inhibitor DIDS. The possible function of VDAC in the synaptic plasma membrane is discussed.     

Incorporation of Acetate into Acetylcholine, Acetylcarnitine, and Amino Acids in the Torpedo Electric Organ

The metabolism of acetate was investigated in the nerve-electroplaque system of Torpedo marmorata. In intact fragments of electric organ, radiolabeled acetate was incorporated into acetylcholine (ACh), acetylcarnitine (ACar), and three amino acids: aspartate, glutamate, and glutamine. These compounds were identified by TLC, high-voltage electrophoresis, column chromatography, and enzymic tests. The system responsible for acetate transport and incorporation into ACh displayed a higher affinity but a lower Vmax than that involved in the synthesis of ACar and amino acids. Choline, when added to the medium, increased the rate of acetate incorporation into ACh but decreased (at concentrations greater than 10(-5) M) that into ACar and amino acids. Monofluoroacetate slightly depressed ACh and ACar synthesis from external acetate but inhibited much more the synthesis of amino acids. During repetitive nerve stimulation, the level of the newly synthetized [14C]ACh was found to oscillate together with that of endogenous ACh, but the level of neither [14C]ACar nor the 14C-labeled amino acids exhibited any significant change as a function of time. This means that there is probably no periodic transfer of acetyl groups between ACh and the investigated metabolites in the course of activity. Acetate metabolism was also tested in the electric lobe (which contains the cell bodies of the neurons innervating the electric organ) and in Torpedo synaptosomes (which are nerve terminals isolated from the same neurons). Radioactive pyruvate and glutamine were also assayed in some experiments for comparison with acetate. These observations are discussed in connection with ACh metabolism under resting and active conditions in tissues where acetate is the preferred precursor of the neurotransmitter.     

12-O-Tetradecanoylphorbol 13-Acetate and Forskolin Modify Muscarinic Receptor-Linked Ca2+ Mobilization in SH-SY5Y Neuroblastoma Cells Through Different Mechanisms

The phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA), which causes differentiation of SH-SY5Y neuroblastoma cells, reduces carbachol binding and carbachol-stimulated Ca2+ mobilization in these cells. The decrease in responsiveness to carbachol is due partially to a reduction in the amount of Ca2+ released by the cells and partially to a decrease in the sensitivity of the cells to carbachol. These effects probably can be attributed to a reduction in muscarinic receptor number and a decrease in receptor affinity, respectively. Forskolin, an alkaloid known to cause an increase in cellular cyclic AMP, enhances Ca2+ influx into the cells without affecting the cytosolic free Ca2+ concentration. The alkaloid causes an apparent restoration of the reduced Ca2+ release, caused by TPA, but does not affect the sensitivity of the cells to carbachol. Forskolin increases the decay of carbachol-induced increase in cytosolic Ca2+. The effects of TPA appear to be linked directly to receptor function, whereas those of forskolin are due to the effect of cyclic AMP on cellular Ca2+ metabolism.     

Purification and Characterization of a Nonvesicular Vesamicol-Binding Protein from Electric Organ and Demonstration of a Related Protein in Mammalian Brain

A protein that binds vesamicol has been purified from a soluble fraction of the Torpedo electric organ homogenate that does not contain synaptic vesicles. The purified vesamicol-binding protein (VBP) has a molecular mass of 470 kDa composed of 30- and 24-kDa subunits. Chemical deglycosylation yielded a single, heterogeneous protein of 24 kDa. The 30-kDa subunit is also sensitive to endo-beta-galactosidase. The dissociation constant of the VBP.vesamicol complex is 0.9 microM, and the Bmax is 5,500 pmol/mg. Antiserum raised to the 30-kDa subunit cross-reacts with the 24-kDa subunit, but not with synaptic vesicles. Drug binding studies and Western blot analysis show that VBP is present in other Torpedo tissues as well as mammalian brain. Immunofluorescence microscopy demonstrates that VBP-like immunoreactivity is not localized exclusively to the nerve terminal regions of the electric organ. Thermal stability, the pH dependence of vesamicol binding, and pharmacological comparisons demonstrate that the VBP is not the cholinergic synaptic vesicle receptor for vesamicol. The implications of this finding for current efforts to develop in vivo diagnostics of cholinergic nerve terminal status based on vesamicol are discussed.