Increased Acetylcholine Content Induced by Adenosine in a Sympathetic Ganglion and Its Subsequent Mobilization by Electrical Stimulation

Abstract: The present study was initiated to examine the effects of ATP on acetylcholine (ACh) synthesis. The exposure of superior cervical ganglia to ATP increased ACh stores by 25%, but this effect was also evident with ADP, AMP, and adenosine, but not with βγ-methylene ATP, a nonhydrolyzable analogue of ATP, or with inosine, the deaminated product of adenosine. Thus, we attribute the enhanced ACh content caused by ATP to the presence of adenosine derived from its hydrolysis by 5′-nucleotidase. The adenosine-induced increase of tissue ACh was not the consequence of an adenosine-induced decrease of ACh release. The extra ACh remained in the tissue for more than 15 min after the removal of adenosine, but it was not apparent when ganglia were exposed to adenosine in a Ca²⁺-free medium. Incorporation of radiolabelled choline into [³H]ACh was also enhanced in the presence of adenosine, suggesting an extracellular source of precursor. Moreover, the synthesis of radiolabelled forms of phosphorylcholine and phospholipid was not reduced in adenosine's presence, suggesting that the extra ACh was not likely derived from choline destined for phospholipid synthesis. Aminophylline did not prevent the adenosine effect to increase ACh content; this effect was blocked by dipyridamole, but not by nitrobenzylthioinosine (NBTI). In addition, two benzodiazepine stereoisomers known to inhibit stereoselectively the NBTI-resistant nucleoside transporter displayed a similar stereoselective ability to block the effect of adenosine. Together, these results argue that adenosine is transported through an NBTI-resistant nucleoside transporter to exert an effect on ACh synthesis. The extra ACh accumulated as a result of adenosine's action was releasable during subsequent preganglionic nerve stimulation, but not in the presence of vesamicol, a vesicular ACh transporter inhibitor. We conclude that the mobilization of ACh is enhanced as a result of adenosine pretreatment.     

Mobilization of a Vesamicol-Insensitive Pool of Acetylcholine from a Sympathetic Ganglion by Ouabain

Abstract: These experiments investigate the release of transmitter from the perfused superior cervical ganglia of cats induced by ouabain in the absence or presence of 2-(4-phenylpiperidino)cyclohexanol (vesamicol), a blocker of acetylcholine (ACh) uptake. Ouabain, perfused through the ganglia, released ACh in a Ca²⁺-dependent way. Vesamicol caused some inhibition of the release of ACh by ouabain; however, under this condition, the Na⁺, K⁺-ATPase inhibitor released five times more transmitter than did preganglionic stimulation at 5 Hz. Also, when ganglia exposed to vesamicol were depleted of the impulse-releasable pool of ACh, subsequent perfusion with ouabain released ACh, and this included ACh newly synthesized in the presence of vesamicol; this phenomenon could be inhibited by the lack of Ca²⁺ and presence of EGTA, and was completely abolished by perfusion with a medium containing 18 mM Mg²⁺. To test whether the release of this vesamicol-insensitive Ca²⁺-dependent pool by ouabain is associated with a decrease in the number of synaptic vesicles, ganglia treated with the ATPase inhibitor after the depletion of the impulse-releasable pool of ACh were fixed for electron microscopy. In the presence of Ca²⁺, coincident with the release of the vesamicol-insensitive pool of ACh, nerve terminals were almost depleted of synaptic vesicles; ganglia treated similarly, but with medium containing 18 mM Mg²⁺ instead of Ca²⁺, were not depleted of synaptic vesicles. These results suggest that ouabain releases a vesamicol-insensitive pool of ACh from the sympathetic ganglion and also support the notion that this compartment is vesicular and its exocytosis depends on extracellular Ca²⁺. It is suggested that empty-vesicle recycling in the presence of vesamicol restricts mobilization of full vesicles to release sites.     

A prolonged after-effect of intense synaptic activity on acetylcholine in a sympathetic ganglion.

The findings was confirmed that there is a "rebound" increase of stored acetylcholine (ACh) in cat superior cervical ganglia conditioned by prolonged preganglionic stimulation at a frequency high enough to cause initial depletion of the store. Ganglia removed immediately after 60 min of continuous or interrupted stimulation at 50 Hz, with chloralose as anesthetic, contained about 30% more ACh than their unconditioned controls; the rebound rose to about 60% after 15 min of rest and then subsided with an apparent half-time of about 2 h. Tests with hemicholinium, combined with hexamethonium or tubocurarine, showed that rebound ACh was located presynaptically and could be released by nerve impulses; but conditioned ganglia perfused with an eserine-containing medium did not release more ACh than their unconditioned controls, except in circumstances in which the mobilization of ACh from a reserve store appeared to be the rate-limiting process for release. The appearance of rebound ACh during and after conditioning stimulation was suppressed by hexamethonium and by tubocurarine, neither of which has much effect on ACh turnover in ganglia excited at lower frequencies, but not only by atropine, noradrenaline, or phenoxybenzamine. The formation of rebound ACH is thus contingent on the postsynaptic nicotinic response to released ACh, and may represent an augmentation of the transmitter store in structures remote from the release sites.     

ACETYLTRIETHYLCHOLINE: A CHOLINERGIC FALSE TRANSMITTER IN CAT SUPERIOR CERVICAL GANGLION AND RAT CEREBRAL CORTEX

The accumulation and metabolism of [¹⁴C]triethylcholine by cat superior cervical ganglia [rested or stimulated (20 Hz)] and by rat cerebral cortex minces was measured. In ganglia, preganglionic nerve stimulation increased the accumulation (2.4 fold) and the acetylation (5.7 fold) of triethylcholine; however the depletion of the ganglion's acetylcholine content was 9.5 times greater than the amount of acetyltriethylcholine synthesized. In the presence of eserine, neither stimulated nor rested ganglia synthesized any extra (surplus) acetyltriethylcholine. It is concluded that the rate-limiting step in acetyltriethylcholine synthesis is the acetylation of triethylcholine by choline acetyltransferase. Subsequent preganglionic nerve stimulation of ganglia, which had been stimulated during the exposure to [¹⁴C]-triethylcholine, caused the increased release of only acetyltriethylcholine; the release was frequency-dependent, required the presence of Ca²⁺, and was blocked by increasing the ratio of Mg²⁺/Ca²⁺ in the perfusion fluid. All of the acetyltriethylcholine which had been accumulated was available for release. Rat cerebral cortex also accumulated triethylcholine and acetylated about 3% of the accumulated choline analogue. Subsequent stimulation by high K⁺ (46 mM)-atropine (3 μM) caused the increased release of acetyltriethylcholine from the cortex and this release required the presence of Ca²⁺. Triethylcholine can therefore form a cholinergic false transmitter in the cat superior cervical ganglion and the rat cerebral cortex.     

Distribution of 45Ca in the superior cervical ganglion of the cat

Superior cervical ganglia isolated from immature cats accumulated 0.9 ng atoms of ⁴⁵Ca per mg wet weight during 10-min incubations at 37°C; when expressed as an equivalent volume of medium the accumulation was four times the uptake of ³H-inulin. Orthodromic stimulation of the ganglia doubled ⁴⁵Ca accumulation, whereas excitation with 50 mM KCl, 5 mM glutamate, or antidromic stimulation increased accumulation by one-half. Hexamethonium reduced the increment in ⁴⁵Ca accumulation due to orthodromic stimulation only, but another ganglionic blocking agent, tetraethylammonium, did not reduce accumulation in any case. Both agents blocked ganglionic transmission monitored electrophysiologically. To resolve this discrepancy, and to approach the localization of ⁴⁵Ca within the ganglia, the efflux of previously accumulated ⁴⁵Ca was examined. The data could be fitted by an equation incorporating the sum of three exponentials, representing a rapidly exchanging compartment plus two more slowly exchanging ones. The latter two appeared to reflect the pre- and postganglionic elements in the ganglia: ⁴⁵Ca content of the “preganglionic” compartment was increased by orthodromic but not by antidromic stimulation, and was not decreased by either blocking agent; conversely, ⁴⁵Ca content of the “postganglionic” compartment was increased by both orthodromic and antidromic stimulation, and was decreased by both blocking agents after orthodromic stimulation. The lack of effect of tetraethylammonium on the whole ganglion resulted from an increase in “preganglionic” accumulation that offset the “postganglionic” decrease. After preganglionic denervation, the ⁴⁵Ca content of the “preganglionic” compartment was reduced by two-thirds, while the ⁴⁵Ca content of the “postganglionic” compartment was unchanged. Chemical stimulation increased ⁴⁵Ca accumulation in both compartments. Diphenylhydantoin, 0.1 mM, decreased the increment in ⁴⁵Ca accumulation due to electrical stimulation and to 50 m^M KCl; this inhibition occurred in the “preganglionic” compartment (and perhaps also in the “postganglionic”), and was accompanied by an increased efflux of ⁴⁵Ca.     

Effects of Colchicine Application to Preganglionic Axons on Choline Acetyltransferase Activity and Acetylcholine Content and Release in the Superior Cervical Ganglion

Abstract: These experiments investigate the effect of block, by colchicine, of fast axonal transport in the cat's cervical sympathetic trunk (CST) on the superior cervical ganglion's choline acetyltransferase (ChAT) enzyme activity, acetylcholine (ACh) content, and ACh release. Electron microscopy on the segment of the CST exposed to colchicine 1 or 4 days earlier showed disappearance of microtubules and accumulation of vesicles and smooth membrane tubules but no disruption of the axonal cytomatrix. At 4 days following colchicine treatment, the number and size of synaptic boutons per grid square in the ganglion ipsilateral to the colchicine-treated CST were similar to those in the control ganglion. At 2 and 4 days following exposure of the CST to colchicine, ChAT activity in the ipsilateral ganglion was reduced to 76 ± 8 and 54 ± 6% of control values, respectively. ACh stores in the ganglia were also reduced (to 81 ± 6% of control values at 2 days and to 51 ± 5% of control values at 4 days). Ganglionic transmission and its sensitivity to blockade by hexamethonium during 2-Hz CST stimulation were not impaired at day 4 postcolchicine. ACh release evoked by 2-Hz stimulation of colchicine-treated axons was similar to release from untreated axons, despite the decrease in the ganglionic ACh content. In contrast, ACh release evoked by 20-Hz stimulation was depressed. The amount of ACh released during 5-Hz stimulation in the presence of vesamicol by the terminals of colchicine-treated axons was similar to that released by the terminals of untreated axons. These results suggest the following conclusions: (a) Colchicine-sensitive fast axonal transport contributes significantly to maintaining ChAT stores in preganglionic axon terminals. (b) The half-life of ChAT in sympathetic preganglionic terminals is ～4 days. (c) One consequence of colchicine-induced block of axonal transport is a reduced ACh content of preganglionic nerve terminals. (d) This decrease in ACh content appears to be the result of a loss in a reserve transmitter pool, whereas the size of the readily releasable compartment is maintained.     

Increased acetylcholine synthesis and release following presynaptic activity in a sympathetic ganglion

Abstract: The acetylcholine (ACh) content of sympathetic ganglia increases above its normal level following a period of preganglionic nerve stimulation. In the present experiments, this extra ACh that accumulates following activity was labeled radioactively from [³H]choline and its specific activity was compared with that of ACh subsequently released during preganglionic nerve stimulation. The specific activity of the released ACh was similar to that of the total tissue ACh, suggesting that the extra ACh mixes fully with endogenous stores. The present experiments also show that transmitter release during neuronal stimulation is necessary for the poststimulation increase in transmitter store. However, the increase was not evident when transmitter release was induced by K⁺. It is concluded that both transmitter release and impulse invasion of the nerve terminals are necessary for the adaptive phenomenon to manifest itself. The role of choline delivery and choline acetyltransferase activity in generating the poststimulation increase in transmitter store was tested. When choline transport activity measured as choline analogue (homocholine) accumulation increased, ACh synthesis was increased and when transport activity was not increased, neither was ACh synthesis. There was no poststimulation increase in measured choline acetyltransferase activity.     

Attenuation of the responses to repeated cholinergic interventions in the isolated dog atrium

In the isolated, blood-perfused canine right atrium, which was pretreated with propranolol, negative chronotropic and inotropic responses were evoked by stimulation of the intramural parasympathetic nerve fibers or by intra-arterial infusion of acetylcholine (ACh). Successive cholinergic interventions were applied; first, a conditioning intervention for 2 min was given, then this was followed by a test intervention for 4 min. The two interventions were separated by a rest period that varied from 15 to 240 s. The cardiac responses to the conditioning parasympathetic nerve stimulation quickly reached maximum levels, and then they "faded" or progressively diminished back toward the control level. The inotropic responses to the conditioning infusion of ACh (1 microgram/min) faded slightly but the chronotropic response did not. After the rest period, the test nerve stimulation evoked responses that also gradually faded with time. The maximal amplitude of the responses to the test stimuli were less than those to the conditioning stimuli. This reduction in the maximal amplitude of the cardiac responses to the test stimuli was more pronounced with high frequency stimulation (30 Hz) than with low frequency stimulation (5 Hz). The decrement was also more pronounced the shorter the rest period, and it was greater at earlier times after beginning the stimulation. Conversely, the maximal cardiac responses to test infusions of ACh were not appreciably less than the responses to the conditioning infusions. We conclude, therefore, that the diminution of the cardiac responses to the second test stimulation of the parasympathetic nerve fibers was mainly ascribable to a prejunctional rather than to a postjunctional mechanism.     

Effects of various kinds of stimulation on ornithine decarboxylase activity in superior cervical sympathetic and nodose ganglia of rats

Levels of cyclic nucleotides and ornithine decarboxylase (ODC) activity were examined following the application of various kinds of stimuli to superior cervical sympathetic ganglia (SCG), nodose ganglia, and vagus nerve fibers excised from the rat. The level of cyclic GMP in the SCG rose rapidly to about 4.5- to 7.5-fold the unstimulated control with 10 min of incubation after applications of preganglionic electrical stimulation (10 Hz), acetylcholine (ACh; 1 mM), or high extracellular K+ ( [K+]0, 70 mM). The cyclic GMP level in nodose ganglia was increased less than in the SCG by either ACh or high [K+]0 but was not affected by ACh in vagus fibers. Cyclic AMP in the SCG was also increased about 4- to 5.5-fold over the control within 10 min with the addition of ACh, norepinephrine (NE; 0.05 mM), or high [K+]0. Although NE caused a small increase in cyclic AMP, neither ACh nor high [K+]0 produced any appreciable change in nodose ganglia or vagus fibers. The ODC activity in the SCG was increased by preganglionic stimulation of 3- to 4-hr duration but not by a shorter period. A similar change in ODC activity was caused by the addition of oxotremorine (1 mM), isoproterenol (0.1 mM), NE, cyclic AMP (1 mM), or dibutyryl cyclic GMP (1 mM). The effect was exaggerated by the further addition of 3-isobutyl-1-methylxanthine (IBMX), a phosphodiesterase inhibitor. The increase in ODC activity caused by ACh was abolished by a muscarinic cholinergic antagonist, atropine (0.01 mM), and following axotomy for a week, but not by a nicotinic antagonist or by denervation in the SCG. A similar increase in ganglionic ODC activity by NE was inhibited by an adrenergic blocker, propranolol (0.01 mM), and following axtotomy for a week, but not by denervation. Cholinergic or adrenergic stimulation did not cause an increase in ODC activity in nodose ganglia or vagus fibers. These results suggest that the stimulation-induced increase in ODC activity occurs in postganglionic neurons rather than in satellite glial cells and is mediated by muscarinic cholinergic or adrenergic receptors. The process appears to involve cyclic nucleotide-mediated protein biosynthesis in the SCG.     

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)     

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 effect of atropine upon acetylcholine release from cat superior cervical ganglia and rat cortical slices: measurement by a radio-enzymic method.

Atropine is known to increase the release of acetylcholine (ACh) from cerebral cortex, and the present experiments tested the effect of this drug upon ACh release in the superior cervical ganglion of the cat. The release of ACh was measured by a radio-enzymic method, which was shown to provide an estimate of the ACh content of samples collected from perfused ganglia that was similar (102%) to that obtained by the method of bioassay more usually used . Atropine (3 X 10(-6) M) increased (3.5 to 4-fold) the amount of ACh released by rat's sliced cerebral cortex incubated in a high (23 mM) potassium medium. However atropine (3 X 10(-6)-3 X 10(-5) M) did not change the amount of ACh released by ganglia during preganglionic nerve stimulation (5-10 Hz). It is concluded that cholinergic nerve terminals in different tissues appear to have different pharmacological properties.     

Storage and release of labelled acetylcholine in the myenteric plexus of the guinea-pig ileum.

Guinea-pig ileum myenteric plexus-longitudinal muscle preparation was superfused with [3H]choline for 15 min either without being stimulated or during field stimulation at 0.1 or 16 Hz; the preparation was then either removed immediately or after 75- or 135-min superfusion with hemicholinium-3 (HC-3) and the total acetylcholine (ACh) and [3H]ACh contents were determined. For measuring the release of [3H]ACh the preparation was stimulated for 60 min the second time at 0.1 or 16 HZ in the presence of hemicholinium. Exposure to [3H]choline without stimulation resulted in the formation of [3H]ACh stores which were maintained in the first 75 min but decreased therafter. Labelling during stimulation at 16 Hz produced the largest and best maintained [3H]ACh content. Following labelling during 0.1-Hz stimulation, more label could be released than following labelling in the absence of stimulation. Labelling during 16-Hz stimulation did not increase any further in fool of [3H]ACh accessible to release by 0.1-Hz stimulation, but caused a 2.5 times increase in the pool from which Hz stimulation released [3H]ACh. These results suggest that two populations of cholinergic neurons exist in the myenteric plexus, one activated only by high frequency stimulation, the other by both high and low frequency stimulation.     

Effects of electrical stimulation and choline availability on the release and contents of acetylcholine and choline in superfused slices from rat striatum

The presence of 5 or 20 microM choline in the eserinized medium superfusing striatal slices enhanced the spontaneous release of acetylcholine (ACh) at both concentrations and, at 20 microM, the release of transmitter evoked by electrical field stimulation. Neither the electrical stimulation nor the addition of choline altered choline acetyltransferase activity. These results show that ACh release is dependent on the availability of extracellular choline. The rate of choline efflux was 7 times higher than the rate of ACh release, was not affected by stimulation, and was increased by 40% when hemicholinium-3 (HC-3), an inhibition of choline uptake, was present. The muscarinic antagonist atropine (1 microM) increased the evoked release of ACh into both the choline-free medium and that containing 20 microM choline. An adenosine receptor antagonist, 1,3-diethyl-8-phenyl xanthine (10 microM), failed to affect ACh release or the enhancement of release produced by atropine. In medium containing HC-3, stimulation of the slices elicited ACh release for the first 20 min of the 30 min stimulation period (15 Hz); thereafter, although stimulation was continued, the rate of release decreased to that associated with spontaneous release. Tissue ACh contents were not modified by the addition of choline or atropine to the medium, but were depressed by HC-3. Neither atropine nor HC-3 altered tissue choline content. The total amount of ACh + choline released during an experiment was 5-15 times higher than the decrease in tissue levels of these two compounds during the same period of time.(ABSTRACT TRUNCATED AT 250 WORDS)     

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)     

Changes in cytoplasmic and vesicular acetylcholine content in insect ganglia during tetanic stimulation

Acetylcholine (ACh) content was reduced by about 30 pmol or 20% of the initial ACh content in the cockroach sixth abdominal ganglion in response to prolonged (30 min) tetanic stimulation at 40 Hz of the cercal nerves in the presence of 10⁻³ M hemicholinium-3 (HC-3). The reduction in ACh content in ganglia occurred in the cytoplasmic rather than the vesicular ACh fraction. The latter showed instead a transient increase followed by a gradual decrease to the previous level. Similar changes in ACh in the fractions were produced also by the stimulation, although the ACh content in ganglia did not change in a calcium-free saline, but was reduced in the presence of 50 μM dantrolene or 1–5 mM cobalt chloride. Synaptic transmission at the cercal nerve-giant nerve fiber synapses rapidly decreased and was abolished within a few minutes during tetanic stimulation at 40 Hz, but recovered on reducing the frequency to 0.1 Hz. The decline in transmission was not affected by HC-3, but a significant delay was observed in the recovery following 30 min of tetanic stimulation in the presence of HC-3.These results may suggest that the depletion of ACh as a functional store occurs in the cytoplasmic ACh fraction, rather than in the vesicular one, after prolonged stimulation in the presence of HC-3. The latter fraction shows and increase in the uptake of cytoplasmic ACh that depend on the presence of intracellular calcium ions during stimulation.     

Synthesis of Acetylcholine from Acetate in a Sympathetic Ganglion

Abstract: The present experiments tested whether acetate plays a role in the provision of acetyl-CoA for acetylcholine synthesis in the cat's superior cervical ganglion. Labeled acetylcholine was identified in extracts of ganglia that had been perfused for 20 min with Krebs solution containing choline (10⁻⁵ M ) and [³H], [1-⁴C], or [2-¹⁴C]acetate (10³ M ); perfusion for 60 min or with [³H]acetate (10⁻² M ) increased the labeling. The acetylcholine synthesized from acetate was available for release by a Ca²⁺-dependent mechanism during subsequent periods of preganglionic nerve stimulation. When ganglia were stimulated via their preganglionic nerves or by exposure to 46 m M K⁺, the labeling of acetylcholine from [³H]acetate was reduced when compared with resting ganglia. The reduced synthesis of acetylcholine from acetate during stimulation was not due to acetate recapture, shunting of acetate into lipid synthesis, or the transmitter release process itself. In ganglia perfused with [2-¹⁴C]glucose, the amount of labeled acetylcholine formed was clearly enhanced during stimulation. An increase in acetylcholine labeling from [³H]acetate was shown during a 15-min resting period following a 60-min period of preganglionic nerve stimulation (20 Hz). It is concluded that acetate is not the main physiological acetyl precursor for acetylcholine synthesis in this sympathetic ganglion, and that during preganglionic nerve stimulation there is enhanced delivery of acetyl-CoA to choline acetyltransferase from a source other than acetate.     

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)     

Nicotinic Autoreceptors Mediating Enhancement of Acetylcholine Release Become Operative in Conditions of "Impaired" Cholinergic Presynaptic Function

Abstract: The existence in the mammalian CNS of release-inhibiting muscarinic autoreceptors is well established. In contrast, few reports have focused on nicotinic autoreceptors mediating enhancement of acetylcholine (ACh) release. Moreover, it is unclear under what conditions the function of one type of autoreceptor prevails over that of the other. Rat cerebrocortex slices, prelabeled with [³H]choline, were stimulated electrically at 3 or 0.1 Hz. The release of [³H]ACh evoked at both frequencies was inhibited by oxotremorine, a muscarinic receptor agonist, and stimulated by atropine, a muscarinic antagonist. Nicotine, ineffective at 3 Hz, enhanced [³H]ACh release at 0.1 Hz; mecamylamine, a nicotinic antagonist, had no effect at 3 Hz but inhibited [³H]ACh release at 0.1 Hz. The cholinesterase inhibitor neostigmine decreased [³H]ACh release at 3 Hz but not at 0.1 Hz; in the presence of atropine, neostigmine potentiated [³H]ACh release, an effect blocked by mecamylamine. In synaptosomes depolarized with 15 mM KCI, ACh inhibited [³H]ACh release; this inhibition was reversed to an enhancement when the external [Ca²⁺] was lowered. The same occurred when, at 1.2 mM Ca²⁺, external [K⁺] was decreased. Oxotremorine still inhibited [³H]ACh release at 0.1 mM Ca²⁺. When muscarinic receptors were inactivated with atropine, the K⁺ (15 mM)-evoked release of [³H]ACh (at 0.1 mM Ca²⁺) was potently enhanced by ACh acting at nicotinic receptors (EC₅₀? 0.6 µM). In conclusion, synaptic ACh concentration does not seem to determine whether muscarinic or nicotinic autoreceptors are activated. Although muscarinic autoreceptors prevail under normal conditions, nicotinic autoreceptors appear to become responsive to endogenous ACh and to exogenous nicotinic agents under conditions mimicking impairment of ACh release. Our data may explain in part the reported efficacy of cholinesterase inhibitors (and nicotinic agonists) in Alzheimer's disease.     

Effect of Subtype-Specific Ca2+-Antagonists and Ca2+-Free Media on the Field Stimulation-Evoked Release of ATP and [3H]Acetylcholine from Rat Habenula Slices

The involvement of different subtypes of voltage-sensitive Ca²⁺ channels in the initiation of field stimulation-induced endogenous adenosine triphosphate (ATP) and [³H]acetylcholine ([³H]ACh) release was investigated in the superfused rat habenula slices. ATP, measured by the luciferin-luciferase assay, and [³H]ACh were released simultaneously from the tissue in response to low frequency electrical stimulation (2 Hz, 2.5 msec, 360 shocks). The N-type Ca²⁺ channel blocker -conotoxin GVIA (-CgTX, 0.01–1 M) reduced the stimulation-evoked release of ATP and [³H]ACh in a dose-dependent manner. Similarly, the P-type Ca²⁺ channel antagonist -agatoxin IVA (-Aga IVA) (0.05 M) and the inorganic Ca²⁺ channel blocker Cd²⁺ (0.2 mM) inhibited the outflow of both transmitters, while Ni²⁺ (0.1 mM) was without significant effect. A high correlation was observed between the percent inhibition of ATP release and percent inhibition of ACh release caused by the different Ca²⁺ antagonists. Long-term perfusion (i.e., 90 min) with Ca²⁺ free solution inhibited the evoked-release of ATP and [³H]ACh. In contrast, perfusion of slices with the same media for a shorter time (i.e., 20 min) did not reduce the release of [³H]ACh and ATP but even increased the evoked-release of ATP about fourfold. The breakdown of extracellular ATP was not blocked under low [Ca²⁺]₀ condition, measured by the creatine phosphokinase assay and HPLC-UV technique. Application of extra- or intracellular Ca²⁺ chelators, and dipyridamole (2 M), the nucleoside transporter inhibitor, did not reduce the excess release of ATP after short-term perfusion with Ca²⁺-free media. Tetrodotoxin (TTX, 1 M), while inhibiting the majority of ATP release under normal conditions, was also unable to reduce release under low [Ca²⁺]₀ conditions. In summary, we showed that both N- and P-type Ca²⁺ channels are involved in the initiation of electrical stimulation-evoked release of ATP and [³H]ACh in the rat habenula under normal extracellular calcium concentration. Under low [Ca²⁺]₀ conditions an additional release of ATP occurs, which is not associated with action potential propagation.