Ganglionic Acetylcholinesterase Research Articles

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The following four proposed roles for the acetylcholinesterase (AChE) of the superior cervical ganglion, where the localization of the enzyme is predominantly presynaptic, were studied: (1) limitation, temporally or spatially, of the transmitter action of acetylcholine (ACh) at the postsynaptic site, (2) hydrolysis of liberated ACh to provide an immediate source of choline for uptake and resynthesis to ACh by the terminals of the preganglionic fibers, (3) protection of the presynaptic nerve terminals against endogenously liberated ACh, and (4) prevention of the accumulation and action of ACh liberated during the resting state. Some of the findings reported previously by others which form the basis for the first proposal were confirmed. However, experiments designed to obtain more definitive evidence for this proposal and, particularly, to assess the degree of the intraganglionic spread of ACh were unsuccessful. No evidence was obtained to support the second proposal. Although the rate of failure of transmission produced by repetitive preganglionic stimulation was accelerated markedly by diisopropyl phosphorofluoridate (isoflurophate, DFP), the infusion of choline, over a wide range of concentrations, produced no antagonism of the failure of transmission either before or after DFP. Thus, it is suggested that the failure of transmission under these conditions was due to the accumulation of excessive concentrations of ACh rather than a temporary depletion of transmitter substance. Support for the third proposal was indicated by the findings that: (a) the mean threshold doses, for postganglionic activation, of carbachol were significantly (26-fold) higher in chronically denervated than in normal ganglia, and (b) although the mean threshold doses of ACh were the same in untreated denervated and normal ganglia, after DFP the thresholds were significantly (5-fold) lower in the latter as compared with the former. The mean threshold dose of ACh was 10 times that of carbachol in normal ganglia; however, in the three situations where AChE was inactivated or absent (<i>i.e.,</i> denervated ganglia before and after DFP, and normal ganglia after DFP), the mean threshold doses of ACh were only one-third to one-half those of carbachol. A limited number of similar experiments was conducted with butyrylcholine and tetramethylammonium. It is inferred from these observations that in normally innervated ganglia, threshold doses of carbachol, and possibly of ACh, activate only the presynaptic terminals, causing them in turn to liberate the neurohumoral transmitter, with consequent activation of the postsynaptic site. Hence, it is suggested that the primary role of ganglionic AChE is the limitation of the action of liberated ACh on the presynaptic nerve terminals themselves. Other possible interpretations are also considered. It was observed also that in denervated ganglia the mean threshold doses of both ACh and carbachol were reduced by DFP to the same extent. The possibilities that the butyrocholinesterase of the capsular glial cells, or some nonspecific group at the receptor site, plays a role in the potentiation of these agents by DFP are discussed. The finding that persistent asynchronous postganglionic firing occurred in acutely decentralized resting ganglia following the injection of larger doses of DFP provides support for the last proposal. Postganglionic firing following massive doses of DFP did not occur in chronically denervated ganglia.

Properties of acetylcholinesterase and non-specific cholinesterase in rat superior cervical ganglion and plasma.

Amphiphile dependency, solubility in aqueous solutions, and sensitivity to proteolysis of acetylcholinesterase (AChE) and nonspecific cholinesterase (nsChE) in the rat superior cervical ganglion were studied and compared to properties of soluble plasma cholinesterases. Ganglion AChE shows strong amphiphile dependency: an amphyphilic substance must be present in the homogenizing medium in order to obtain maximal apparent enzyme activity. Apparent activity of AChE solubilized in Ringer's solution was also increased after subsequent addition of a detergent. The 4 S molecular form, predominant in this extract (corresponding to the fastest electrophoretic band), is very sensitive to papain proteolysis but can be protected by a detergent. This molecular form therefore carries an important hydrophobic domain and is probably membrane bound in situ. The 10 S form of ganglionic AChE, extracted in Ringer's solution, is probably a soluble enzyme since, like soluble plasma enzymes, it is not amphiphile dependent and is rather resistant to proteolysis. Ganglion nsChE is more water soluble, less amphiphile dependent and more protease resistant than AChE.

Noncholinesterase actions of an irreversible acetylcholinesterase inhibitor on synaptic transmission and membrane properties in autonomic ganglia.

Superfusion of the organophosphorous acetylcholinesterase inhibitor soman (pinacolyl methylphosphonofluoridate; 0.01-25 microM) produced a dose-dependent reduction of extracellularly and intracellularly recorded synaptic responses in the isolated rat superior cervical ganglia at frequencies of orthodromic stimulation that do not normally produce synaptic depression. The magnitude of depression was dependent upon the frequency of stimulation (0.02-1 Hz), was maintained after the removal of soman from the superfusion solution, and recovered by over 65% during periods of inactivity. The depression of synaptic transmission produced by soman was not dependent upon the inhibition of acetylcholinesterase (AChE) activity by this agent. Transmission was increasingly depressed by doses of soman greater than those needed to inactivate all measurable ganglionic AChE activity. Dose-dependent depression of synaptic transmission in soman also occurred after pretreatment with the irreversible AChE inhibitor diisopropylphosphofluoridate (DFP; 100 microM), which inhibited greater than 98% of the AChE activity in the ganglia. Soman produced a decline in the input resistance, resting potential, spike amplitude, and spike threshold and a reduction in the hyperpolarizing afterpotential. Soman-induced depression of synaptic transmission was not due primarily to a blockade of postsynaptic nicotinic receptors. At concentrations of soman which produced significant depression in transmission, ganglionic depolarization produced by bath-applied carbamylcholine (carbachol) was either slightly depressed or facilitated. In the presence of soman, repetitive focal application of acetylcholine or carbachol did not reveal use-dependent desensitization. Muscarinic antagonists, atropine and pirenzepine, protected against the use-dependent depression of synaptic transmission induced by soman. These results suggest that a principal site of action for soman is at the presynaptic terminal and that this site is sensitive to muscarinic receptor blockade.

Acetylcholinesterase localization at synapses in chick embryo ciliary ganglion.

The cytochemical localization of acetylcholinesterase (AChE) at calyciform synapses of the chick ciliary ganglion during embryonic development has been investigated. AChE activity is present at the surface membrane of newly formed calyciform synapses and closely follows the progressive enlargement of the synaptic area. The occurrence of a retrograde iris-dependent influence on ganglionic AChE is considered. AChE seems to be a suitable marker for synaptic maturation.

Demonstration of a neurotrophic factor for the maintenance of acetylcholinesterase and butyrylcholinesterase in the preganglionically denervated superior cervical ganglion of the cat.

Under sodium pentobarbital anesthesia, the superior cervical ganglia of cats were preganglionically denervated bilaterally. The following day cats were reanesthetized, the external carotid and lingual arteries were ligated bilaterally, and the right common carotid artery was infused for 24 hr with an extract prepared from cat brain, spinal cord, and sciatic nerves, with and without the incorporation of aprotinin, an inhibitor of proteases. They were sacrificed 48 hr after denervation, and the acetylcholinesterase (acetylcholine acetylhydrolase, EC 3.1.1.7) and butyrylcholinesterase (acylcholine acylhydrolase, EC 3.1.1.8) contents of the superior cervical ganglia were compared with those of similarly denervated control ganglia. Both types of extract produced a significant reduction in the loss of both enzymes from the superior cervical ganglia, as did infusions of aprotinin alone. These findings demonstrate the presence of an endogenous neurotrophic factor for the maintenance of ganglionic acetylcholinesterase and butyrylcholinesterase. Its possible mechanisms of action, and those of aprotinin, are discussed.

Multiple molecular forms of rat superior cervical ganglion acetylcholinesterase: Developmental aspects in primary cell culture and during postnatal maturation in vivo

Four main molecular forms of acetylcholinesterase (AChE) can be solubilized from newborn rat superior cervical ganglia (SCG), homogenized in the presence of a high-ionic-strength, detergent-containing medium. These forms, respectively referred to as 16, 10, 6.5, and 4 S, are characterized by their sedimentation coefficients. Their relative proportions in SCG are notably different in vivo during postnatal maturation, and in culture. The 16-S AChE appears to be mainly neuronal in origin, is maintained in culture independently of original presynaptic in vivo elements, and its cellular pool is not depleted in the presence of tetrodotoxin (TTX).

Effects of selective alkylphosphorylation of propionylcholinesterase on the regeneration of acetylcholinesterase in the aqueous soluble fraction of superior cervical ganglia of the rat following sarin.

The rates of regeneration of acetylcholinesterase (AChE) and propionylcholinesterase (PrChE) in the supernatants of aqueous homogenates of rat superior cervical ganglia, centrifuged at 100,000 g for 90 min, were determined at 1, 3, 6, and 16 h following their inactivation (greater than 90%) by administration of sarin, 2.0 mumol/kg i.v. Values were compared with those in animals in which the PrChE was continually suppressed by the repeated, fractional administration of iso-OMPA, in a total dose of 10 or 20 mumol/kg i.p. These doses of iso-OMPA alone produced 96-99% inactivation of PrChE with no detectable effect on AChE. Significant suppression of AChE regeneration by iso-OMPA administration was noted only at 6 h; in contrast with earlier findings in the cat, administration of iso-OMPA alone caused no significant increase in ganglionic AChE activity.

Acetylcholinesterase molecular forms in chick ciliary ganglion: pre- and postsynaptic distribution derived from denervation, axotomy, and double section.

Abstract: Four main molecular forms of acetylcholinesterase (AChE) characterized by their sedimentation coefficients (5S, 7.5S, 11.5S, and 20S), are found in chick ciliary ganglion. After transection of the preganglionic nerve (denervation), total AChE activity in the ganglion dropped by 35% in 2 days. By then, 11.5s and 20s forms had diminished by 60 and 75% respectively, where as 7.5s remained practically unchanged. Since presynaptic structures disappeared 2 days after denervation, we inferred that at most 35% of total ganglion AChE was presynaptic: 11.5s and 20s might be mainly presynaptic and 7.5S, postsynaptic. At later time intervals. total AChE continued to decline up to day 5, possibly as a result of orthograde transynaptic regulation of the enzyme activity. After transection of postganglionic nerves (axotomy), total ganglion activity showed little change; 11.5s and 20s decreased by 40 and 6076, respectively, in 5 days, but these drops were compensated for by an early increase in 7 5S, which started the day after axotomy. After simultaneous transection of both pre‐ and postganglionic nerves (double section), total ganglion AChE dropped rapidly by 35% in 1 day and remained at that level up to 21 days. The 11.5S diminished rapidly by 60% in 1 day. The early increase of the 7.5s form induced by axotomy alone did not occur. Since the effect resulting from double section was not the equivalent of the cumulative effects observed after denervation and axotomy, respectively, the level of AChE forms in the ganglion may be regulated by reciprocal interaction of pre‐ and postsynaptic elements. After denervation and double section but not after axotomy alone, the contralateral non‐operated ganglion exhibited a fall in the 20s form. This suggests that a transynaptic effect is exerted on AChE by the contralateral preganglionic neuron. Taken together, these results indicate that the various AChE molecular forms in chick ciliary ganglion are preferentially but not exclusively distributed as follows: the pre‐ and postganglionic axons contain mainly the 11.5S form, whereas nerve endings and synaptic structures are enriched in 20S, and ganglion cell bodies, in 7.5s.

Development of the multiple molecular forms of acetyl-cholinesterase in chick paravertebral sympathetic ganglia: An in vivo and in vitro study

The development of acetylcholinesterase (AChE) activity and the distribution of this enzyme among its multiple forms was studied in both tissue extracts and dissociated cell cultures of chick paravertebral sympathetic ganglia. In agreement with previous findings, total AChE (expressed either per ganglion or per μg protein) increased in vivo between the time of formation of the paravertebral chain (embryonic day 7; E7) to hatching (E20-E21). After this time, enzyme activity changed much more slowly. Sucrose gradient sedimentation analysis of AChE in ganglia of post-hatching chicks revealed multiple forms of AChE with S values of approximately 6.5, 11 and 19.5 Developmental studies showed that 6.5 S and 11 S forms are present as early as day E7. Much of the pre-hatching increase in total AChE is due to increased levels of the 6.5 S form of the enzyme. By hatching, this form comprised approximately 85–90% of the total AChE activity. In contrast, during the first week after hatching, the activity of the 11 S form increased several-fold while that of the 6.5 S remained approximately unchanged. The 19.5 S form, which is thought to be associated with the synaptic membrane, was not detected prior to day E17 and reached adult levels (2–3% of total AChE activity) by the first week after hatching. Development of AChE was also studied in dissociated cell cultures of embryonic ganglia. Essentially all the AChE activity in such cultures was found to be associated with the neurons. Total AChE activity of cultured E11 ganglia increased in a pattern which was both qualitatively and quantitatively similar to that which occured in vivol. Furthermore, it was found that development of both the 6.5 and 11 S forms of AChE took place in vitro. In cultures of E8, E11, E15, and E19 ganglia, the distribution of activity between the two forms after various times in vitro was similar to that which was found for in vivo ganglia at an equivalent embryonic stage. Such changes were not affected by the elimination of non-neuronal cells from the cultures. Two aspects of in vitro development, however, differed from that which occured in vivo. First, an increase in 11 S AChE did not occur at ages equivalent to the first week post-hatching. Second, the 19.5 S form did not develop (even after several weeks) in cultures of E8, E11 and E15 ganglia, nor was this form (which was removed during dissociation of the ganglia) regenerated in cultures of E19 ganglia. Such findings suggest that the pattern of development of AChE and its multiple forms in chick sympathetic neurons is in part intrinsically programmed into these cells at an early stage of development as well as in part regulated by extrinsic signals that these cells receive from their chemical and cellular environment.

STEADY STATE AND REGENERATING LEVELS OF ACETYLCHOLINESTERASE IN THE SUPERIOR CERVICAL GANGLION OF THE RAT FOLLOWING SELECTIVE INACTIVATION OF PROPIONYLCHOLINESTERASE1

Abstract— The effects of selective inactivation of propionylcholinesterase (PrChE) by tetramonoisopropylpyrophosphortetramide (iso‐OMPA) on the steady state and regenerating levels of acetylcholinesterase (AChE) were investigated on the superior cervical ganglion (SCG) of the rat. Over the dosage range of 1.5‐40.0 μmol iso‐OMPA/kg intraperitoneally, which produced nearly total inactivation of ganglionic PrChE and 0‐35% inactivation of AChE, there was no subsequent increase in AChE activity above the control level. Single or repeated injections of iso‐OMPA at total doses of 5.0‐40.0 μmol/kg intraperitoneally caused no reduction in the rate of regeneration of ganglionic AChE during the 24 h following its inactivation by sarin, 2.0 μmol/kg intravenously. Both sets of findings differ from those obtained previously in a similar study of ganglionic AChE and butyrylcholinesterase (BuChE) in the cat. Possible reasons for this distinct species difference are discussed.

Interrelationships between ganglionic acetylcholinesterase and nonspecific cholinesterase of the cat and rat.

When homogenates of cat or rat superior cervical ganglia in Krebs-Ringer solution were incubated at 37 degrees C, the ensuing decrease in acetylcholinesterase (acetylcholine acylhydrolase, EC 3.1.1.7) activity was increased significantly by prior administration in vivo of tetramonoisopropylpyrophosphotetramide at doses that produced selective alkylphosphorylation of butyrylcholinesterase or propionylcholinesterase. These findings are consistent with the proposal that the latter enzymes are posttranscriptional precursors of acetylcholinesterase. Results of similar studies with homogenates of ganglia in water or in M NaCl/1% Triton X-100 were inconclusive, as were those of heat-inactivation studies and immunoprecipitation of the enzymes.

Effects of inactivation of butyrylcholinesterase on steady state and regenerating levels of ganglionic acetylcholinesterase.

Abstract—The effects of single and repeated injections of tetramonoisopropyl pyrophosphortetramide (iso‐OMPA), a selective inactivator of butyrylcholinesterase (BuChE), were studied on the ganglionic and muscular levels of BuChE and acetylcholinesterase (AChE) in cats during the steady state and following the irreversible inactivation of both enzymes by isopropylmethylphosphonofluoridate (sarin). Single intravenous injections of iso‐OMPA, 3.0 or 6.0 μmol/kg, produced nearly total inactivation of BuChE with no immediate effect on the AChE of the superior cervical (SCG), stellate (StG), and ciliary (CG) ganglia and inferior oblique (10) muscle; regeneration of BuChE occurred at approximately the same rate in the three ganglia, and at 4–6 days the AChE levels were significantly elevated. When single doses of iso‐OMPA were given 1 h following sarin, 2.0 μmol/kg, intravenously, there was a slight increase in the rate of AChE regeneration during the ensuing 2 days. With the repeated injection of iso‐OMPA, 3.0 μmol/kg every 48 h, there was a consistent but not statistically significant reduction in AChE regeneration at 4, 6, 12, and 18 days following sarin in all 3 ganglia. Similar treatment with iso‐OMPA alone produced significant increases in ganglionic AChE at all these periods excepting the longest. The daily injection of iso‐OMPA for 6 days, which maintained ganglionic BuChE at approx 2% of the control values, produced significant reductions in AChE regeneration, but again significant increases in ganglionic AChE levels in cats that did not receive sarin. The IO muscle did not exhibit these effects. A working hypothesis is proposed, that BuChE is a precursor of ganglionic AChE, and that the level of BuChE participates in the regulation of AChE synthesis by inhibition of a preceding rate‐limiting step.

Effect of preganglionic application of vinblastine-impregnated silastic cuffs on acetylcholinesterase, butyrocholinesterase, and acetylcholine contents of cat superior cervical ganglion

Vinblastine sulfate in silastic cuffs was applied to the preganglionic vagosympathetic trunk of the cat superior cervical ganglion in an attempt to assess the role of acetylcholine as a possible neurotrophic factor required for maintaining postsynaptic ganglionic acetylcholinesterase and butyrocholinesterase. Following implantation of 1% (w/w) vinblastine cuffs, specific activities of acetylcholinesterase and butyrocholinesterase in the ganglion decreased to approximately 20 and 50% of control activities, respectively, within 1 week. The acetylcholine content of the superior cervical ganglion of most cats showed a moderate to marked decrease within this period as well. Histochemical findings were indistinguishable from those seen following preganglionic denervation. Because of the similar fall in ganglionic acetylcholine and enzyme contents, results with this technique do not eliminate acetylcholine as a possible neurotrophic factor for the maintenance of postsynaptic ganglionic acetyl- and butyrocholinesterases.

Effects of persistent selective suppression of ganglionic butyrylcholinesterase on steady state and regenerating levels of acetylcholinesterase: implications regarding function of butyrylcholinesterase and regulation of protein synthesis.

Persistent selective suppression of the butyrylcholinesterase (cholinesterase; acylcholine acyl-hydrolase, EC 3.1.1.8) activity of the superior cervical, stellate, and ciliary ganglia of cats by the daily administration of tetramonoisopropyl pyrophosphortetramide, 3.0 mumol/kg, intravenously, for 6 days produced a significant elevation in the levels of ganglionic acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7). When the same procedure was preceded by the inactivation of over 95% of the ganglionic acetylcholinesterase by sarin, 2.0 mumol/kg, intravenously, the rate of regeneration of acetylcholinesterase was decreased. Results are interpreted as evidence that ganglionic butyrylcholinesterase may serve as a precursor to acetylcholinesterase, and that the level of butyrylcholinesterase may regulate the rate of acetylcholinesterase synthesis.

Responses of normal and denervated cat superior cervical ganglia to some stimulant compounds.

1. The effect of preganglionic denervation on the sensitivity of the cat superior cervical ganglion to close-arterial injections of some stimulant compounds in vivo has been studied.2. Ganglionic responses were assessed (a) by recording simultaneously ganglion depolarization and post-ganglionic nerve action potential discharges, and (b) by measuring the contractions of the nictitating membrane and applying a correction for the change in sensitivity of the membrane to noradrenaline.3. No change could be detected in the electrical responses of the ganglion to carbachol after denervation.4. Nictitating membrane recording suggested that denervation increased the response of the ganglion to acetylcholine, but had little effect on ganglionic sensitivities to carbachol, nicotine, tetramethylammonium or potassium chloride.5. It was concluded that the neurones of the superior cervical ganglion do not, in general, show any substantial change of sensitivity to cholinesterase-resistant stimulant compounds after denervation. The supersensitivity to acetylcholine is probably the result of a reduction of ganglionic acetylcholinesterase.

Cholinesterases of rat sympathetic ganglia after immunosympathectomy, decentralization and axotomy.

Abstract— Immunosympathectomy was produced in Sprague‐Dawley rats by the subcutaneous injection of 300 units of nerve growth factor (NGF)‐antiserum (1.56 mg of freeze‐dried serum)/g/day for 6 days, the first dose being given 5–8 hr after birth. The immunosympathectomized rats and their control littermates were killed 2½ and 7 months after birth.Ganglionic acetylcholinesterase and pseudocholinesterase activities were measured by an adaption (Kungman, Kungman and Pouszczuk, 1968) of the colorimetric method of Ellman, Courtney, Andres and Featherstone (1961). Following immunosympathectomy the activities of these enzymes decreased significantly in superior cervical, stellate, thoracic chain, cardiac (abdominal), coeliac and superior mesenteric ganglia. The reduction of the acetylcholinesterase activity was greater than expected in a number of sympathetic ganglia, e.g. superior cervical, stellate, coeliac and cardiac ganglia, if one considered that only the postganglionic neurons were affected by immunosympathectomy. The activities of these enzymes were also reduced in the cervical sympathetic trunks from NGF‐antiserum‐treated rats. By means of decentralization and axotomy it was shown that 45 per cent of the total ganglionic acetylcholinesterase activity was associated with the preganglionic and 55 per cent with the postganglionic elements of the superior cervical ganglion from control rats. It was concluded that immunosympathectomy also affects the preganglionic sympathetic neurons. It is not known whether this is a primary effect of the NGF‐antiserum or a secondary effect resulting from the absence of over 90 per cent of the postganglionic sympathetic cell bodies.

The cytological localization of intracellular neuronal acetylcholinesterase.

Sections of cat ciliary ganglia were stained for acetylcholinesterase activity by several modifications of the acetylthiocholine method in order to achieve optimal accuracy of cytological localization of the enzyme. These were compared by ordinary light and phase contrast microscopy with similar sections stained by standard techniques for Nissl substance, the Golgi apparatus, and the neurofibrillae, and by intravital methylene blue. The pattern of cytoplasmic distribution of acetylcholinesterase corresponded most closely with that of the Nissl substance. Following total inactivation of the ganglionic acetylcholinesterase by intravenously administered di-isopropyl fluorophosphate, the reappearance of the enzyme in vivo occurred at the same cytoplasmic sites prior to its reappearance at the cell membrane or preganglionic axonal terminations. These observations, and reports cited from the literature, provide support for the hypothesis that acetylcholinesterase is synthesized within the endoplasmic reticulum, then transported via its canaliculi to the surface of the cell and its processes, where its functional sites are oriented externally to the lipoidal membrane.