THE ACCUMULATION OF RADIOACTIVE ACETYLCHOLINE BY A SYMPATHETIC GANGLION AND BY BRAIN: FAILURE TO LABEL ENDOGENOUS STORES

Abstract— The accumulation of radioactively labelled acetylcholine (ACh) by perfused superior cervical ganglia of cats and by incubated brain slices from rats was studied in the presence of diisopropylphosphorofluoridate. Ganglia accumulated more labelled ACh than an extracellular marker (inulin), but the amount of ACh accumulated did not increase when ACh turnover was increased by preganglionic nerve stimulation. The ACh that accumulated in ganglia was not released when the preganglionic nerve was subsequently stimulated. Sliced cerebral cortex also accumulated labelled ACh but this was not released when the tissue was subsequently exposed to a high K⁺ medium. Thus accumulated ACh does not appear to mix with releasable transmitter stores. Chronically (7 days) decentralized ganglia lost most of their transmitter store but retained their ability to accumulate labelled ACh. Uptake of ACh by sliced cerebellum was not less than uptake of ACh by sliced cerebral cortex and the amount of ACh accumulated by synaptosomes isolated from cerebellum was similar to the amount of ACh accumulated by synaptosomes isolated from cerebral cortex. It is concluded that ACh uptake is not specifically into cholinergic nerve endings. Hexamethonium reduced ACh uptake by cerebral cortex slices but did not increase the amount of ACh collected from slices stimulated by raised K⁺.     

The site of the neuromuscular block produced by polymyxin B and rolitetracycline.

The site of neuromuscular blockade induced by polymyxin B and rolitetracycline was studied on isolated nerve and nerve-muscle preparations. Polymyxin B (1.8 X 10(-4) M) was equipotent to lidocaine as a local anaesthetic on a frog desheathed nerve preparation, while rolitetracycline (up to 3.6 X 10(-3)M) had no local anaesthetic effect. Polymyxin B (6 X 10(-5) M) and rolitetracycline (7 X 10(-4) M) blocked by 50% the response of rat diaphragm induced by phrenic nerve stimulation, but did not decrease the amount of acetylcholine (ACh) released from this preparation during nerve stimulation. Both antibiotics depressed the response of the rat diaphragm to inject ACh, and this response was more sensitive to inhibition by the drugs than was the response to nerve stimulation. With rolitetracycline, a concentration that blocked the response to nerve stimulation by 50% inhibited the response to injected ACh by 85%, and this relationship was similar to that with d-tubocurarine; however, polymyxin B was relatively more effective than d-tubocurarine in inhibiting the effect of ACh. Polymyxin B (1-1.5 X 10(-4) M) but not rolitetracycline (1 X 10(-3) M) depressed the response of the diaphragm to direct muscle stimulation. It is concluded that polymyxin B and rolitetracycline block neuromuscular transmission predominatly by an effect to depress the muscle's sensitivity to ACh; polymyxin B probably acts by an effect similar to that of local anaesthetics, while rolitetracycline probably acts by an effect similar to that of d-tubocurarine.     

Effects of Nucleus Basalis Lesions on the Muscarinic and Nicotinic Modulation of [3H] Acetylcholine Release in the Rat Cerebral Cortex

Presynaptic muscarinic and nicotinic receptors in the cerebral cortex reportedly inhibit and increase acetylcholine (ACh) release, respectively. In this study, we investigated whether these receptors reside on cholinergic nerve terminals projecting to the cerebral cortex from the nucleus basalis magnocellularis (nbm). Adult male rats received unilateral infusions of ibotenic acid (5 micrograms/1 microliter) in the nbm. Two weeks later, cerebral cortical cholinergic markers (choline acetyltransferase activity, high-affinity choline uptake, and coupled ACh synthesis) were significantly reduced in synaptosomes prepared from the lesioned hemispheres compared to contralateral controls. The depolarization-induced release of [3H]ACh from these synaptosomes was also reduced in the lesioned hemispheres, reflecting the reduced synthesis of transmitter. However, the nbm lesions had no effect on the inhibition of release induced by 100 microM oxotremorine. Synaptosomal [3H]ACh release was not altered by nicotine or the nicotinic agonists anabaseine and 2-(3-pyridyl)-1,4,5,6-tetrahydropyrimidine. Nicotine (10-100 microM) did increase [3H]ACh release in control and lesioned hemispheres in cortical minces, but to a similar extent. These results suggest that neither muscarinic nor nicotinic receptors modulating ACh release reside on nbm-cholinergic terminals.     

Stimulation of cholinergic receptors and changes in cyclic nucleotide levels in the cerebral cortex of the guinea pig

The effect of Atropine and Physostigmine on 3'5' AMP and 3'5' GMP content was investigated in slices of guinea-pig cerebral cortex maintained at rest or electrically-stimulated. Atropine and Physostigmine did not modify either the basal content or the electrically-evoked increase of 3'5' AMP and 3'5' GMP. On the contrary, Betanechol 25 micro M significantly increased 3'5' GMP and 3'5' AMP content in slices kept at rest. The effect was abolished by Atropine 1,5 x 10(-7) M and d-tubocurarine 7 x 10(-6) M, respectively.     

Acetylcholine Mobilization in a Sympathetic Ganglion in the Presence and Absence of 2-(4-Phenylpiperidino)Cyclohexanol (AH5183)

The present experiments measured the release of acetylcholine (ACh) by the cat superior cervical ganglia in the presence of, and after exposure to, 2-(4-phenylpiperidino)cyclohexanol (AH5183), a compound known to block the uptake of ACh by cholinergic synaptic vesicles. We confirmed that AH5183 blocks evoked ACh release during preganglionic nerve stimulation when approximately 13-14% of the initial ganglial ACh stores had been released; periods of rest in the presence of the drug did not promote recovery from the block, but ACh release recovered following the washout of AH5183. ACh was synthesized in AH5183-treated ganglia, as determined by the synthesis of [3H]ACh from [3H]choline, and this [3H]ACh could be released by stimulation following drug washout. The specific activity of the released ACh matched that of the tissue's ACh, and thus we conclude that ACh synthesized in the presence of AH5183 is a releasable as pre-existing ACh stores once the drug is removed. We tested the relative releasability of ACh synthesized during AH5183 exposure (perfusion with [3H]choline) and that synthesized during recovery from the drug's effects (perfusion with [14C]choline: the ratio of [3H]ACh to [14C]ACh released by stimulation was similar to the ratio in the tissue. These results suggest that the mobilization of ACh for release by ganglia during recovery from an AH5183-induced block is independent of the conditions under which the ACh was synthesized. Unlike nerve impulses, black widow spider venom (BWSV) induced the release of ACh from AH5183-blocked ganglia, even in the drug's continued presence. Venom-induced release of ACh from AH5183-treated ganglia was not less than the venom-induced release from tissues not exposed to AH5183. This effect of BWSV was attributed to the action of the protein, alpha-latrotoxin, because an anti-alpha-latrotoxin antiserum blocked the venom's action. ACh synthesized during AH5183 exposure was labelled from [3H]choline, and subsequent treatment with BWSV released [3H]ACh with the same temporal pattern as the release of total ACh. To exclude a nonexocytotic origin for the [3H]ACh released by BWSV, ganglia were preloaded with [3H]diethylhomocholine to form [3H]acetyldiethylhomocholine, an ACh analogue excluded from vesicles; the venom did not increase the rate of [3H]acetyldiethylhomocholine efflux. It is concluded that a vesicular ACh pool insensitive to the inhibitory action of AH5183 might exist and that this vesicular pool is not mobilized by electrical stimulation to exocytose in the presence of AH5183, but it is by BWSV.     

Comparative research on synaptic transmission in the sympathetic ganglia of rabbits and frogs during acetylcholinesterase inhibition

Organophosphorus inhibitor of acetylcholinesterase (AChE) armin (1 x 10(-6) M) induced a variety of pre- and postsynaptic effects resulting from the AChE inhibition and subsequent accumulation of acetylcholine (ACh) in the synaptic cleft. The intensity of postsynaptic effects (level of neuron depolarization, degree of action potential depression) was shown to be different in the ganglia of frog and rabbit. This could be explained by differences in the total amount of ACh released in response to nerve stimulation as well as at rest. Both muscarinic and nicotinic cholinoreceptors were involved in the process of sustained depolarization of the neurons in the rabbit superior cervical ganglion after AChE inhibition. In frog ganglion neurons the nicotinic receptors did not participate in depolarization evidently due to their fast desensitization. The activation of presynaptic muscarinic receptors resulted in decrease of ACh released by nerve stimulation seems to weaken depolarization and blockade of synaptic transmission in sympathetic ganglia treated by AChE inhibitors.     

Acetylcholine Synthesis by a Sympathetic Ganglion in the Presence of 2-(4-Phenylpiperidino)cyclohexanol (AH5183) and Picrylsulfonic Acid

The present experiments measured the release and the synthesis of acetylcholine (ACh) by cat sympathetic ganglia in the presence of 2-(4-phenylpiperidino)cyclohexanol (AH5183 or vesamicol) and/or picrylsulfonic acid (TNBS), two compounds known to have the ability to block the uptake of ACh by cholinergic synaptic vesicles in vitro. We confirmed that, in stimulated (5 Hz) perfused (30 min) ganglia, AH5183 depressed ACh release and ACh tissue content increased by 86 +/- 6% compared to contralateral ganglia used as controls. Preganglionic activity increased ACh release by a similar amount in the presence (19.9 +/- 1.0 pmol/min) or absence (20.5 +/- 2.4 pmol/min) of TNBS. The final tissue ACh content was also similar in the presence (1,668 +/- 166 pmol) or absence (1,680 +/- 56 pmol) of TNBS. However, the AH5183-induced increase of tissue ACh content (86 +/- 6%) was abolished completely when AH5183 was perfused with 1.5 mM TNBS (-3.0 +/- 1.0%). This inhibition of ACh synthesis, observed in TNBS-AH5183-perfused ganglia, was not dependent upon further inhibition of ACh release beyond that caused by AH5183 alone, because 14.0 +/- 1.9% of the transmitter store was released by preganglionic nerve stimulation in the presence of TNBS plus AH5183 and this was similar in the presence of AH5183 without TNBS (14.0 +/- 0.6%). Moreover, when ganglia were first treated with TNBS and then stimulated in the presence of AH5183, an increase of 64 +/- 6% of the ganglionic ACh content occurred, and this increase was not statistically different from the increase measured with AH5183 alone (86 +/- 6%).(ABSTRACT TRUNCATED AT 250 WORDS)     

Involvement of prostaglandins in the response of frog adrenocortical cells to muscarinic receptor activation

The role of prostaglandins (PGs) in the mechanism of action of acetylcholine (ACh) on frog adrenocortical cells has been examined. Administration of a single dose of ACh (5 X 10(-5) M) to perifused frog interrenal fragments, for 20 min, stimulated the production of corticosterone, aldosterone, PGE2 and 6-keto-PGF1 alpha. In contrast ACh did not significantly alter TXB2 production. The effect of ACh could be mimicked by muscarine (10(-5) M). Conversely, nicotine (10(-6) to 10(-4) M) was totally inactive. The increase in PG biosynthesis preceded the peak of corticosteroid release. Repeated 20-min pulses of ACh (5 X 10(-5) M) or muscarine (10(-5) M) given at 130-min intervals induced a desensitization phenomenon. In presence of indomethacin (5 X 10(-6) M), the effect of ACh on PG and steroid secretion was totally abolished. In calcium-free medium, the effect of ACh on PG and corticosteroid production was completely blocked. These results indicate that, in the frog, ACh stimulates corticosteroid secretion through a PG-dependent mechanism.     

SYNTHESIS OF RADIOACTIVE ACETYLCHOLINE FROM [3H]CHOLINE AND ITS RELEASE FROM CEREBRAL CORTEX SLICES IN VITRO

Abstract— Guinea pig cerebral cortex slices were incubated for 60 min in a medium containing [³H]choline with or without the addition of 33 mM-KCl for the last 30 min. KC1 caused the release into the medium of large amounts of both bioassayable and radioactive ACh, while at the same time their concentrations in the tissue decreased. The specific activity (d.p.m./pmol) of the ACh released by KC1 was greater than that released in control incubations, indicating that it comes from a newly synthesized, more radioactive store. The amounts of [³H]choline, [³H]ACh and the specific activity of tissue acetylcholine reached a plateau in the tissue 30 min after the addition of isotope. However isotopic equilibrium was not achieved because the specific activity of the ACh released, with or without KC1 in the subsequent 30 min, was less than the specific activity of the ACh remaining in the tissue. This implies the existence of a pool of ACh in the tissue which is turning over very slowly or is being synthesized from a less radioactive pool of choline. This pool of ACh does not contribute substantially to that released by KC1. Levorphanol at 10⁻³ M, as well as the analgesically inactive stereoisomer, dextrorphan, blocked the KCl-stimulated release of both bioassayable and radioactive ACh. These drugs demonstrate the coupling of synthesis and release of ACh in cerebral cortex slices.     

Presynaptic alpha-adrenoceptor mediated inhibition of the neurogenic cholinergic contraction in the isolated intestinal bulb of the carp (Cyprinus carpio)

The effects of norepinephrine, epinephrine and clonidine on neurogenic cholinergic contraction were examined in the presence of a beta-adrenoceptor blocking agent, carteolol (5 X 10(-6) M), in the isolated intestinal bulb of the carp. Norepinephrine, epinephrine (10(-9)-10(-6) M) and clonidine (10(-8)-10(-5) M) inhibited the contraction induced by low frequency (2 or 5 Hz) transmural stimulation (TMS) without inhibiting the contraction induced by acetylcholine (ACh, 6 X 10(-8)-4 X 10(-7) M). Methoxamine (10(-4) M) and phenylephrine (10(-4) M) showed no such inhibitory effect on the TMS-induced contraction. The inhibitory effects of catecholamines and clonidine were decreased by phentolamine (5.4 X 10(-6) M) and yohimbine (10(-7)-10(-6) M) but not by prazosin (7 X 10(-7)-10(-6) M). Nicotine (10(-6)-10(-4) M) and serotonin (3 X 10(-8)-3 X 10(-6) M) caused contraction of the intestinal bulb indirectly by releasing endogenous ACh. This contraction was inhibited by norepinephrine, epinephrine and clonidine in a concentration-dependent manner. The present results suggest that catecholamines and clonidine inhibit cholinergic transmission via the activation of a presynaptic alpha-adrenoceptor (presumably of alpha-2 type) located on the cholinergic nerve terminals innervating the smooth muscle of the intestinal bulb of the carp.     

Regulation of Histamine Release and Synthesis in the Brain by Muscarinic Receptors

The cholinergic modulation of histamine release and synthesis was studied in rat brain slices or synaptosomes labeled with L-[3H]histidine. Carbachol in increasing concentrations progressively reduced the K+-induced [3H]histamine release from cortical slices. Pirenzepine, a preferential M1-receptor antagonist, reversed the carbachol effect in an apparently competitive manner and with Ki values of 1-6 X 10(-8) M. 11-[(2-[(Diethylamino)methyl]-1-piperidinyl)acetyl]-5,11-dihydro-6H- pyrido[2,3-b][1,4]benzodiazepine-6-one (AF-DX 116), considered a preferential M2-receptor antagonist, reversed the carbachol effect with a mean Ki of approximately 2 X 10(-7) M. Oxotremorine behaved as a partial agonist in the modulation of histamine release. Neostigmine, an acetylcholinesterase inhibitor, inhibited the K+-induced release of [3H]histamine from cortical slices, and the effect was largely reversed by pirenzepine, an observation suggesting a modulation by endogenous acetylcholine. The effects of carbachol and pirenzepine were observed with slices of other brain regions known to contain histaminergic nerve terminals or perikarya, as well as with cortical synaptosomes. The two drugs also modified, in opposite directions, [3H]histamine formation in depolarized cortical slices. In vivo oxotremorine inhibited [3H]histamine formation in cerebral cortex, and this effect was reversed by scopolamine. When administered alone, scopolamine failed to enhance significantly the 3H- labeled amine formation, a finding suggesting that muscarinic receptors are not activated by endogenous acetylcholine released under basal conditions. It is concluded that muscarinic heteroreceptors, directly located on histaminergic nerve terminals, control release and synthesis of histamine in the brain. These receptors apparently belong to the broad M1-receptor category and may correspond to a receptor subclass displaying a rather high affinity for AF-DX 116.     

Functional antagonism of D-1 and D-2 dopaminergic mechanisms affecting striatal acetylcholine release

Rat striatal slices prelabelled with [3H]choline were superfused with dopamine D-1 and D-2 agonists and antagonists, separately and in combination, during measurement of [3H]acetylcholine (ACh) release. SKF38393 (D-1 agonist), 10(-7)-10(-4) M, and SCH23390 (D-1 antagonist), 10(-7)-10(-5) M, produced a dose-dependent increase in [3H]ACh release when given separately. The increased [3H]ACh release induced by either drug could not be attenuated by sufficient L-sulpiride to block D-2 receptors. Yet both SKF38393, 10(-6)-10(-5) M, and SCH23390, 10(-6)-10(-5) M, were able to partially or fully overcome the [3H]ACh release-depressant effect of cosuperfused LY171555 (D-2 agonist), 10(-6) M. This suggests that a functional antagonism regarding striatal ACh release exists between D-1 and D-2 dopaminergic receptor-mediated mechanisms, but that D-1 modulation of local ACh release does not occur at the level of the recognition site of the striatal D-2 receptor. Finally, although attenuation of the increased release of striatal [3H]ACh induced by 10(-5) M SCH23390 by SKF38393 was seen, it is possible that such functional antagonism is not mediated by exclusively D-1 dopaminergic means.     

Effect of acetylcholine on ventrocaudal sensory neurons in the pleural ganglia ofAplysia

1. The effects of acetylcholine (ACh) on the soma of cultured ventrocaudal sensory neurons from the pleural ganglia of Aplysia kurodai were characterized. 2. Whole-cell recording was used for current and voltage clamping. ACh and other drugs were microapplied to the membranes of the cultured neurons. 3. Microapplication of ACh induced an outward current mediated by a conductance increase. No desensitization to repeated applications of ACh was detected. The threshold was 10(-7) M and the maximum response was at 10(-5) M. 4. The reversal potential in normal seawater is -80 mV, close to the K+ equilibrium potential. Increasing [K+]0 shifted the reversal potential by the amount predicted by the Nernst equation. Altering [Cl-]0 did not affect the reversal potential. Thus ACh opens a potassium channel in these sensory neurons and may act as a neurotransmitter on those neurons. 5. Atropine and d-tubocurarine partially blocked the ACh response. Hexamethonium had no obvious effect on this response. Tetraethylammonium reduced the response to 22% of control. Carbamylcholine and arecoline induced outward currents that were 71 and 12%, respectively, of the response to ACh. Nicotine and muscarine had almost no effect. 6. The ACh response was reduced by prior application of serotonin (5HT). The ACh response was also reduced by bath-applied 5HT, forskolin, and isobutylmethylxanthine. These data suggest that ACh activates an "S-like" channel in the ventrocaudal sensory neurons.     

Histological and pharmacological investigations of the two symmetrically situated giant neurons, RPeNLN and LPeNLN, identified on the anterior surface of the pedal ganglia of an African giant snail (Achatina fulica Férussac)

Morphological and pharmacological investigations were made of two giant neurons, RPeNLN (right pedal nerve large neuron) and LPeNLN (left pedal nerve large neuron), situated symmetrically on the anterior surface of the pedal ganglia of an African giant snail (Achatina fulica Férussac). The two neurons (about 250-300 microns in diameter) were the largest ones identified in the ganglia of the snail species. The axonal pathways of the two neurons were symmetrical; of their four main axonal branches, the three main branches innervated the ipsilateral pedal nerves, whereas the last main branch projected to the contralateral pedal nerves. The pharmacological features of the two neurons were very similar. Both were inhibited markedly by dopamine [minimum effective concentrations (MECs): 3 X 10(-6)-10(-5) M], DL-octopamine (MECs: 2 X 10(-6)-2 X 10(-5) M), 5-hydroxytryptamine (MEC: 3 X 10(-6) M), GABA (MEC: 3 X 10(-4) M), L-homocysteic acid (MECs: 3 X 10(-5)-10(-4) M) and erythro-beta-hydroxy-L-glutamic acid (MEC: 3 X 10(-5) M). Acetylcholine showed varied effects, either excitatory or inhibitory, on the two neurons examined. No substances were found to have any marked excitatory effects on the neurons.     

Modulation of cholinergic responsiveness through the [beta]-adrenoceptor signal transmission pathway in bovine trachealis.

The effects of pharmacological stimulation at different levels of the beta-adrenoceptor (AR) pathway, including the receptor, the receptor-coupled Gs protein, and adenylyl cyclase, were studied by simultaneous measurements of acetylcholine (ACh) release and isometric force evoked by electric stimulation in isolated bovine trachealis. The beta-AR agonists isoproterenol (10-6 and 10-5 M) and salbutamol (10-7 to 10-5 M) significantly attenuated both ACh release and contractile force. Forskolin, at 10-6 M, significantly increased ACh release without effect on contractile force, whereas at 10-5 M it increased ACh release but significantly decreased force. Activation of Gs protein by cholera toxin (10 microg/ml) significantly attenuated both ACh release and contractile force, but its effect on ACh release was abolished by calcium-activated potassium (KCa)-channel blocker iberiotoxin (10-7 M). The KCa-channel opener NS-1619 (10-4 M) attenuated significantly both ACh release and contractile force. It is concluded that beta-AR agonists attenuate cholinergic neurotransmission in isolated bovine trachealis model by a mechanism not involving cAMP but KCa channels.     

Acetylcholine Synthesis and Release by a Sympathetic Ganglion in the Presence of 2-(4-Phenylpiperidino) Cyclohexanol (AH5183)

These experiments measured the release and the synthesis of acetylcholine (ACh) by cat sympathetic ganglia in the presence of 2-(4-phenylpiperidino) cyclohexanol (AH5183), an agent that blocks the uptake of ACh into synaptic vesicles. Evoked transmitter release during short periods of preganglionic nerve stimulation was not affected by AH5183, but release during prolonged stimulation was not maintained in the drug's presence, whereas it was in the drug's absence. The amount of ACh releasable by nerve impulses in the presence of AH5183 was 194 +/- 10 pmol, which represented 14 +/- 1% of the tissue ACh store. The effect of AH5183 on ACh release was not well antagonized by 4-aminopyridine (4-AP), and not associated with inhibition of stimulation-induced calcium accumulation by nerve terminals. It is concluded that AH5183 blocks ACh release indirectly, and that the proportion of stored ACh releasable in the compound's presence represents transmitter in synaptic vesicles available to the release mechanism. The synthesis of ACh during 30 min preganglionic stimulation in the presence of AH5183 was 2,448 +/- 51 pmol and in its absence it was 2,547 +/- 273 pmol. Thus, as the drug decreased ACh release it increased tissue content. The increase in tissue content of ACh in the presence of AH5183 was not evident in resting ganglia; it was evident in stimulated ganglia whether or not tissue cholinesterase was inhibited; it was increased by 4-AP and reduced by divalent cation changes expected to decrease calcium influx during nerve terminal depolarization.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regional Selectivity of a γ-Aminobutyric Acid-Induced [3H]Acetylcholine Release Sensitive to Inhibitors of γ-Aminobutyric Acid Uptake

The effects of gamma-aminobutyric acid (GABA) on the release of [3H]acetylcholine ([3H]ACh) were studied in synaptosomes prepared from rat hippocampus, cerebral cortex, hypothalamus, and striatum and prelabelled with [3H]choline. When synaptosomes were exposed in superfusion to exogenous GABA (0.01-0.3 mM) the basal release of newly synthesized [3H]ACh was increased in a concentration-dependent way in hippocampus, cortex, and hypothalamus nerve endings. In contrast, the release of [3H]ACh was not significantly affected by GABA in striatal synaptosomes. The effect of GABA was not antagonized significantly by bicuculline or picrotoxin. Muscimol caused only a slight not significant increase of [3H]ACh release when tested at 0.3 mM whereas, at this concentration, (-)-baclofen was totally inactive. The GABA-induced release of [3H]ACh was counteracted by SKF 89976A, SKF 100561, and SKF 100330A, three strong and selective GABA uptake inhibitors. The data suggest that, in selective areas of the rat brain, GABA causes release of [3H]ACh following penetration into cholinergic nerve terminals through a GABA transport system.     

Mobilization of the Readily Releasable Pool of Acetylcholine from a Sympathetic Ganglion by Tityustoxin in the Presence of Vesamicol

The present experiments tested whether preganglionic stimulation and direct depolarization of nerve terminals by tityustoxin could mobilize similar or different pools of acetylcholine (ACh) from the cat superior cervical ganglia in the presence of 2-(4-phenylpiperidino)cyclohexanol (vesamicol, AH5183), an inhibitor of ACh uptake into synaptic vesicles. In the absence of vesamicol, both nerve stimulation and tityustoxin increased ACh release. In the presence of vesamicol, the release of ACh induced by tityustoxin was inhibited, and just 16% of the initial tissue content could be released, a result similar to that obtained with electrical stimulation under the same condition. When the impulse-releasable pool of ACh had been depleted, tityustoxin still could release transmitter, amounting to some 10% of the ganglion's initial content. This pool of transmitter seemed to be preformed in the synaptic vesicles, rather than synthesized in response to stimuli, as tityustoxin could not release newly synthesized [3H]ACh formed in the presence of vesamicol, and hemicholinium-3 did not prevent the toxin-induced release. In contrast to the results with tityustoxin, preganglionic stimulation could not release transmitter when impulse-releasable or toxin-releasable compartments had been depleted. Our results confirm that vesamicol inhibits the mobilization of transmitter from a reserve to a more readily releasable pool, and they also suggest that, under these experimental conditions, there might be some futile transmitter mobilization, apparently to sites other than nerve terminal active zones.     

Accumulation, Acetylation, and Releasability of Diethylhomocholine from a Sympathetic Ganglion

Superior cervical ganglia of the cat perfused with [14C]diethylhomocholine [( 14C]DEHCh) synthesized acetyldiethylhomocholine (ADEHCh), but rather little of this ester was released by subsequent preganglionic nerve stimulation. Stimulation evoked the release of an appreciable amount of unchanged DEHCh when ganglia had been exposed to the analogue in the absence of choline (Ch), but did not do so when exposed to both Ch and DEHCh. The release of DEHCh was Ca2+ dependent, and was not the result of the release and subsequent hydrolysis of ADEHCh. This is the first clear demonstration of the release of an unacetylated compound from mammalian tissue; therefore, the characteristics of the transmitter release mechanism are further defined. The effect of preganglionic nerve stimulation on the uptake and acetylation of DEHCh was also measured. Stimulated ganglia accumulated approximately 4 times more labeled analogue and synthesized 7.5 times more ADEHCh than did rested ganglia. Stimulated ganglia perfused with 2-(4-phenylpiperidino)cyclohexanol, a compound considered to inhibit acetylcholine (ACh) release by inhibiting its transport into synaptic vesicles, accumulated 3.4 times as much and acetylated 6 times as much DEHCh as did rested ganglia. When the concentration of Mg2+ in the perfusion medium was increased to block ACh release, accumulation of the labelled analogue was enhanced by stimulation, but its acetylation was increased much less than during perfusion with normal medium. It is concluded that the synthesis of ADEHCh is subject to the same regulation as is ACh synthesis and that the activation of ester synthesis during activity can be dissociated from ester release.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pharmacologic evidence for direct dopaminergic regulation of striatal acetylcholine release

This study was done to provide pharmacologic evidence for the location of those striatal dopamine D-1 and D-2 receptors that participate in the regulation of local acetylcholine (ACh) release. Striatal tissue slices from adult male Sprague-Dawley rats were preloaded with [3H]choline and superfused in separate experiments with buffer containing either: a D-2-specific agonist (LY141865 or LY171555), a D-2 specific antagonist (L-sulpiride), a D-1 specific agonist (SKF38393), or a D-1 antagonist (SCH23390), in the presence or absence of tetrodotoxin (TTX), used to block interneuronal activity. With either D-2 agonist there was a dose-dependent decrease in K+-stimulated [3H]ACh release, maximally at 5 X 10(-7)-10(-6) M [agonist] and to the same extent with each drug. Both SKF38393 and SCH23390 increased [3H]ACh release at tested concentrations of these agents. Results were unchanged when any of the drugs used was superfused in the presence of TTX, 5 X 10(-7) M. These data are consistent with the hypothesis that populations of striatal D-1 and D-2 receptors exist on local cholinergic neurons, where they regulate ACh release. Alternative interpretations are discussed.