The effect of soman on potassium evoked 3H-acetylcholine release in the isolated rat bronchi

The in vitro exposure of rat bronchial smooth muscle to the acetylcholinesterase inhibitor soman (0-[1,1,2-trimethylpropyl]-methylphosphonofluoridate) reduced the potassium (51 mM) evoked release of 3H-acetylcholine (3H-ACh). Exposure to 1.0 and 100 microM soman for 15 min inhibited the acetylcholinesterase (AChE) activity completely and reduced the potassium evoked release by 23.1% and 34.4% respectively. In the presence of scopolamine (0.3 microM), however, there was a large enhancement (87.0%) of potassium evoked release during soman inhibited (100%) AChE-activity. Furthermore, soman (1.0 microM) did not reduce the spontaneous release of 3H-ACh. The results indicate that the presynaptic effect of soman is due to the enhanced concentration of ACh following AChE-activity inhibition in the synaptic region. This induces a stimulation of presynaptic muscarinic receptors and thereby modulation of the ACh release only during evoked release.     

Butyrylcholinesterase and acetylcholinesterase activity and quantal transmitter release at normal and acetylcholinesterase knockout mouse neuromuscular junctions

1 The present study was performed to evaluate the presence and the physiological consequences of butyrylcholinesterase (BChE) inhibition on isolated phrenic-hemidiaphragm preparations from normal mice expressing acetylcholinesterase (AChE) and BChE, and from AChE-knockout mice (AChE(-/-)) expressing only BChE. 2 Histochemical and enzymatic assays revealed abundance of AChE and BChE in normal mature neuromuscular junctions (NMJs). 3 In normal NMJs, in which release was reduced by low Ca(2+)/high Mg(2+) medium BChE inhibition with tetraisopropylpyrophosphoramide (iso-OMPA) or bambuterol decreased ( approximately 50%) evoked quantal release, while inhibition of AChE with fasciculin-1, galanthamine (10, 20 micro M) or neostigmine (0.1-1 micro M) increased (50-80%) evoked quantal release. Inhibition of both AChE and BChE with galanthamine (80 micro M), neostigmine (3-10 micro M), O-ethylS-2-(diisopropylamino)ethyl-methylphosphono-thioate (MTP) or phospholine decreased evoked transmitter release (20-50%). 4 In AChE(-/-) NMJs, iso-OMPA pre-treatment decreased evoked release. 5 Muscarinic toxin-3 decreased evoked release in both AChE(-/-) and normal NMJs treated with low concentrations of neostigmine, galanthamine or fasciculin-1, but had no effect in normal NMJs pretreated with iso-OMPA, bambuterol, MTP and phospholine. 6 In normal and AChE(-/-) NMJs pretreatment with iso-OMPA failed to affect the time course of miniature endplate potentials and full-sized endplate potentials. 7 Overall, our results suggest that inhibition or absence of AChE increases evoked quantal release by involving muscarinic receptors (mAChRs), while BChE inhibition decreases release through direct or indirect mechanisms not involving mAChRs. BChE apparently is not implicated in limiting the duration of acetylcholine action on postsynaptic receptors, but is involved in a presynaptic modulatory step of the release process.     

Cholinergic system of brain tissue in rats poisoned with the organophosphate, 0,0-dimethyl 0-(2,2-dichlorovinyl) phosphate

The cholinergic system of the brain was investigated in rats acutely poisoned with the organophosphate, 0,0-dimethyl 0-(2,2-dichlorovinyl) phosphate (DDVP), (6 mg/kg, sc, with saline as a control). The amounts of three fractions of acetylcholine (ACh)--free (extraterminal), labile-bound (intraterminal/cytoplasmic), and stable-bound (intraterminal/vesicular)--increased in the rats over a period of 5 to 60 min after injection of DDVP, showing peaks which were 2.45, 1.82, and 1.4 times as high as the respective control amounts. No difference was seen in the amount of any fraction of ACh between treated and control rats killed 3 and 24 hr after injection. Acetylcholinesterase (AChE) activity decreased to between 12 and 43% of the control over a period of 5 to 180 min and recovered almost completely within 24 hr after injection. No appreciable changes were seen in either spontaneous or potassium-induced ACh release in brain tissue slices obtained from rats treated with DDVP. ACh synthesis in slices was suppressed significantly 20 min, but not 24 hr, after injection of DDVP. In the brain crude synaptosomal preparation, high-affinity choline uptake, which is generally thought to be a rate-limiting step for ACh synthesis, was suppressed 20 min after DDVP. No appreciable changes were seen in high-affinity choline uptake at 24 hr low-affinity choline uptake, and choline acetyltransferase activity after injection of DDVP. These results suggest that ACh synthesis and high-affinity choline uptake may be in a suppressed state when ACh concentration, especially intraterminal ACh, is increased and AChE activity is decreased in the brain cholinergic system of rats poisoned with DDVP. The increase in the intraterminal ACh may be due to an inhibition of AChE activity at this site and/or a re-uptake of ACh in the synaptic cleft, not to an inhibition of ACh release or an increase in ACh synthesis.     

COMPARATIVE PRESYNAPTIC NEUROCHEMICAL CHANGES IN RAT STRIATUM FOLLOWlNG EXPOSURE TO CHLORPYRIFOS OR PARATHION

Organophosphorus pesticides (OPs) exert acute toxicity through inhibition of acetylcholinesterase (AChE) in target tissues. Previous studies in our laboratory have demonstrated, however, that dosages of the OPs chlorpyrifos (CPF) or parathion (PS), which cause similar degrees of brain AChE inhibition in adult male rats, can produce marked differences in toxicity. While compensatory changes in postsynaptic receptors can modulate the clinical expression of AChE inhibition and lead to tolerance to these toxicants, we propose that OP-selective changes in presynaptic cholinergic processes can also regulate the ultimate consequences of AChE inhibition. The relative effects of either vehicle (peanut oil, 2 ml/ kg, sc), CPF (280 mg/kg), or PS (6.6 mg/ kg) on clinical signs of toxicity and AChE activity, high-affinity choline uptake (HACU), and potassium evoked acetylcholine (ACh) release in striatum were examined for a 7-d period after exposure in adult female Sprague-Dawley rats. In vitro effects of CPF-oxon or paraoxon, the active oxidative metabolites of CPF and PS, on HACU were also examined in comparison with the prototype inhibitor hemicholinium-3 (HC-3) . Similar to our previous findings in male rats, female rats treated with dosages of CPF or PS causing similar maximal degrees of AChE inhibition (82-96%) exhibited marked differences in response; that is, PS produced more extensive signs of acute toxicity (salivation, lacrimation, urination and/or defecation, i.e., SLUD signs and involuntary movements). CPF reduced striatal synaptosomal HACU at 1, 2, and 7 d after exposure, whereas PS only decreased HACU at 2 d posttreatment. While CPF-oxon was a weak inhibitor of HACU (IC50 >200 mu M), paraoxon had no effect on this process in vitro. Potassium-evoked ACh release in the presence of physostigmine (20 mu M) was not affected by either OP at 1 d but was increased 2 d after either CPF or PS treatment and remained elevated at 7 d after exposure in CPF-treated rats only. ACh release in the presence of both physostigmine and the muscarinic antagonist atropine (1 mu M) was decreased by both OPs as early as 1 d after exposure and remained lower at 2 d posttreatment. By 7 d, however, ACh release in response to atropine was decreased in CPF-treated animals only, sug gesting that both CPF and PS affected muscarinic autoreceptor function but with somewhat different time courses. These results suggest that different OPs may selectively modify presynaptic cholinergic processes and that early, OP-selective changes in HACU/ACh synthesis may contribute to the differential toxicity noted following extensive AChE inhibition by either CPF or PS.     

Presynaptic regulation of neurotransmitter release in the cortex of aged rats with differential memory impairments

Cluster analysis of water-maze reference-memory performances of 25-27-month-old (compared to 3-5-month-old) rats distinguished subpopulations of young adult rats (YOUNG), aged rats with no significant impairment (AU), aged rats with moderate impairment (AMI), and aged rats with severe impairment (ASI). In the frontoparietal cortex, we subsequently assessed the electrically evoked release of tritium in slices preloaded with [3H]choline, [3H]noradrenaline (NA), or [3H]serotonin (5-HT) and the effects of an agonist (oxotremorine, UK 14,304, and CP 93,129) of the respective autoreceptors. Cholinergic and monoaminergic markers were measured in homogenates. Overall, aged rats exhibited reduced accumulation of [3H]choline (-25%) and weaker evoked transmitter release (in % of accumulated tritium: -44%, -20%, and -34%, for [3H]acetylcholine, [3H]NA, and [3H]5-HT, respectively). In all rats, the inhibitory effects of the autoreceptor agonists on the evoked release of [3H] were comparable. Acetylcholinesterase (AChE), not choline acetyltransferase (ChAT), activity was reduced. The results suggest age-related modifications in the cholinergic, noradrenergic, and serotonergic innervation of the frontoparietal cortex, alterations of evoked transmitter release, but no interference with presynaptic autoinhibition of the release. Neither of these alterations seemed to account for the cognitive impairment assessed.     

Effects of acetylcholine and atropine on the release of 14C-acetylcholine formed from U-14C-glucose in rat brain cortical and striatal prisms

Summary A perfusion technique has been used to study the mechanisms involved in the control of acetylcholine (ACh) release from rat brain cortical and striatal slices submitted to KCl depolarization. Tissues were superfused continuously with U-¹⁴C-glucose, a very efficient precursor of the acetyl moiety of ACh, and with eserine to prevent the hydrolysis of the ¹⁴C-ACh released in the superfusion medium.This radioenzymatic approach was more flexible than those in which endogenous ACh outflows were estimated using a biological or a radioenzymatic method. Moreover, the modulation of the ester release process could be studied using the physiological ligand of the presynaptic muscarinic receptor, i.e. ACh itself. The continuous synthesis of ¹⁴C-ACh from U-¹⁴C-glucose, throughout the superfusion experiment, allowed the rate of ¹⁴C-ACh release to reach a steady state, under resting conditions (5.6 mM KCl) as well as under conditions of stimulation by high concentrations of KCl. This method was very convenient for pharmacological studies as compared to other methods in which the efflux of ³H-ACh from performed intratissular stores of ³H-ACh was measured.Using this technique, it was shown that atropine (10 M) enhanced the rate of ¹⁴C-ACh spontaneous release. Curiously, extraneously added ACh (0.6 mM) had a similar effect. When cortical prisms were submitted to a brief 31 mM KCl pulse stimulation (3 min), the negative feedback regulation process was not fully triggered. Indeed, atropine, which prevents the interaction of the released ACh with the regulatory muscarinic receptor, significantly stimulated the ¹⁴C-ACh outflow only when brain tissues were submitted to a sustained 31 mM KCl depolarisation, or when the ACh concentration in the superfusion fluid was raised (0.6 mM). These results suggest that the muscarinic negative control of ACh release from brain cholinergic terminals becomes fully operative only when the nerve endings are depolarized and when the ACh concentration within the synapse exceeds a threshold, i.e. during intense functioning of the cholinergic synapse.Abbreviations AChE acetylcholinesterase - ChAc cholineacetyltransferase (EC 2.3.1.6)     

Effects of 2,4-dithiobiuret treatment in rats on cholinergic function and metabolism of the extensor digitorum longus muscle

Effects of 2,4-dithiobiuret (DTB) treatment in rats on neuromuscular transmission and the disposition of cholinergic substances, acetylcholine (ACh) and choline (Ch), were examined in a combined electrophysiological/biochemical study using an in vitro extensor digitorum longus (EDL) muscle-peroneal nerve preparation. EDL muscle preparations isolated from rats treated with DTB (1 mg/kg/day X 5 days, ip) displayed a 49% depression in the frequency of miniature end-plate potentials (MEPPs) and a 21% depression in mean MEPP amplitude. Statistical analysis of evoked end-plate potentials (EPPs) measured in curarized preparations indicated that the mean quantal content (m) was significantly depressed in EDL muscles from DTB-treated rats. At stimulation rates of 1, 10, 20, and 50 Hz the estimated values of m in EDL preparations from DTB-treated rats were, respectively, 21, 25, 45, and 51% of that in control preparations. Biochemical determinations of ACh and Ch revealed a significant DTB-induced increase in endogenous ACh and Ch content in EDL preparations fixed for extraction of ACh and Ch immediately after dissection from the treated rats. In vitro, however, there were negligible changes in overall ACh synthesis since the total (tissue and medium) tracer ACh (2H4-ACh) synthesized from tracer Ch (2H4-Ch; 10 microM) supplied in the perfusion medium was similar in EDL preparations from DTB-treated and control rats. Also, in EDL muscles from DTB-treated rats the resting release of ACh was not affected, but when exogenous Ch (2H4-Ch) was not supplemented in the medium the evoked release (via peroneal nerve stimulation) of ACh was depressed. Thus, decreases in spontaneous quantal ACh release, as detected in the electrophysiological experiments, were not reflected by changes in the biochemically determined ACh resting release. The biochemical determination of evoked ACh release, however, correlated with the decrease in quantal content detected in the electrophysiological analysis of evoked EPPs when exogenous Ch was not supplemented in the perfusion medium. Significant and consistent increases (two to three times) in both Ch content and efflux occurred in the EDL muscles from DTB-intoxicated rats. These results indicate that DTB induces a prejunctional impairment of neuromuscular transmission that is not specifically directed at ACh synthesis. Rather those processes by which ACh is incorporated into or released from vesicles appear to be altered.     

Choline acetyltransferase activity and mass fragmentographic measurement of acetylcholine in specific nuclei and tracts of rat brain.

The mass fragmentographic assay of acetylcholine (ACh) is the only available method to measure the acetylcholine content of rat brain nuclei. The ACh concentration and the choline acetyltransferase activity (ChA) of specific rat brain nuclei and tracts are reported. The highest ACh concentration (1.2 $\text{nmol}\text{mg}$ protein) and ChA activity (576 $\text{nmol}\text{mg}$ protein per hr) was in the nucleus interpeduncularis, which possessed twice as much ACh and ChA as the nucleus accumbens and nucleus caudatus. The midbrain nuclei: dorsalis raphes and linearis pars caudalis contained as much ACh as the nuclei accumbens and caudatus but the ChA activity was only a fraction of that of the accumbens and caudatus. The three pontine nuclei: locus eoeruleus, tegmenti dorsalis and dorsalis vagi contained slightly less ACh than the nuclei accumbens and caudatus but the ChA varied. It was low in the locus coeruleus but 10- fold higher in the dorsalis vagi. It is proposed that the ratio of ACh content to ChA activity may have some predictive value to determine whether most of the ACh measured in various brain nuclei is located in cell bodies or axon terminals. The data presented are compared with histochemical data on the location of acetylcholinesterase (Palkovits and Jacobowitz, 1974). This comparison suggests that when the ratio ACh/ChA × 100 is greater than 0.7 and the ACh content is 0.30 $\text{nmol}\text{mg}$ protein or greater, the transmitter may be located in nerve terminals. When this ratio is smaller than 0.4 and the ChA activity is greater than 5.0 $\text{nmol}\text{mg}$ protein per hr, the ACh measured may be located in cholinergic cell bodies or small cholinergic interneurones. This suggestion is supported by measurement of the ACh concentration and ChA activity in brain nuclei which are known to contain cholinergic cell bodies (e.g. motor nucleus of the vagus), small cholinergic interneurones (n. caudatus and n. accumbens) and cholinergic nerve terminals (n. locus coeruleus, dorsalis raphes).     

Effect of distigmine bromide on the central cholinergic system

AbstractDistigmine bromide (3, 3'-[hexamethylenebis (methyliminocarbonyloxy)] bis (1-methylpyridinium) dibromide), an acetylcholinesterase (AChE) inhibitor, produced a time-dependent and dose-dependent increase in acetylcholine (ACh) levels in the medial prefrontal cortex of rats. The overt cholinergic behaviours, such as tremor, fasciculation and lacrimation, were also elicited by distigmine bromide. The onset and duration of these behaviours were reflected in the microdialysis data showing that distigmine bromide enhances cholinergic neurotransmission in rat brain. The dose of distigmine bromide eliciting increase in ACh in the medial prefrontal cortex of rats correlated well with its dose for the induction of the cholinergic behaviours. Furthermore, distigmine bromide was an equipotent inhibitor of AChE and butyrylcholinesterase (BuChE) activities in the present study. From these findings, it is suggested that distigmine bromide may produce centrally mediated behavioural signs by increasing the ACh levels in the brain, resulting from its AChE and BuChE inhibitions.     

Regional difference of ROS generation, lipid peroxidaton, and antioxidant enzyme activity in rat brain and their dietary modulation

One of the potential causes of age-related neuronal damage can be reactive oxygen species (ROS), as the brain is particularly sensitive to oxidative damage. In the present study, we investigated the effects of aging and dietary restriction (DR) on ROS generation, lipid peroxidation, and antioxidant enzymes in cerebrum, hippocampus, and cerebellum of 6-, 12-, 18-, and 24-month-old rats. ROS generation significantly increased with age in cerebrum of ad libitum (AL) rats. However, no significant age-difference was observed in hippocampus and cerebellum. DR significantly decreased ROS generation in cerebrum and cerebellum at 24-months. On the other hand, the increased lipid peroxidation of AL rats during aging was significantly reduced by DR in all regions. Our results further showed that catalase activity decreased with age in cerebellum of AL rats, which was reversed by DR, although SOD activity had little change by aging and DR in all regions. In a similar way, glutathione (GSH) peroxidase activity increased with age in cerebrum of AL rats, while DR suppressed it at 24-months. These data further support the evidence that the vulnerability to oxidative stress in the brain is region-specific.     

夏天无总生物碱对痴呆大鼠学习记忆障碍及中枢胆碱能神经系统功能的影响

目的　观察夏天无总生物碱 (Xiatianwutotalalkaloids,XA)对喹啉酸损毁海马所致大鼠学习记忆障碍和中枢胆碱能神经系统功能的影响。方法　采用双侧海马CA1区微注射喹啉酸造成大鼠学习记忆障碍模型 ,在造模前 1wk至造模后 3wk内 ,大鼠每天igXA 0 2 5 ,0 5 ,1mg·kg-1。采用三等分Y迷宫测定大鼠学习记忆能力 ,并采用比色法检测海马乙酰胆碱酯酶 (acetylcholinesterase ,AChE)活性和大脑皮层乙酰胆碱 (acetylcholine ,ACh)含量的变化。结果　大鼠双侧海马内微注射喹啉酸可引起大鼠学习记忆功能障碍 ,海马中AChE活性下降 ,大脑皮层ACh含量减少 ;XA可显著改善喹啉酸诱导的痴呆大鼠学习记忆功能障碍 ,同时明显抑制模型大鼠海马的AChE活性 ,提高大脑皮层ACh含量。结论　XA可改善喹啉酸损毁海马造成的痴呆大鼠学习记忆功能障碍 ,而其机制可能与抑制脑AChE活性 ,从而提高ACh含量 ,增强中枢胆碱能系统的功能有关。     

Smooth muscle and parasympathetic nerve terminals in the rat urinary bladder have different subtypes of alpha(1) adrenoceptors

Neurally evoked contractions and release of (3)H- acetylcholine (ACh) during electrical field stimulation were measured in rat urinary bladder strips. The alpha(1) agonist phenylephrine (PE, 2-8 microM) increased the amplitude of neurally evoked contractions, facilitated the release of ACh and increased the baseline tone of the bladder strips. The PE-induced facilitation of the contractions did not significantly change during a prolonged exposure to PE (120 min), whereas the PE-induced rise in baseline tone gradually decreased to 65% of the initial value. Low concentrations of specific alpha(1A) antagonists, 5-methyl urapidil (5-MU), REC15/2739 and WB-4101 competitively inhibited the facilitation of the neurally-evoked contractions (pA(2:) 8.77; 9.59 and 9.62, respectively), whereas higher concentrations of 5-MU (IC(50): 48 nM) were required to suppress the PE-rise in baseline. WB-4101 (100 microM) inhibited the PE-induced facilitation of ACh release. The irreversible alpha(1B) antagonist chloroethyl-clonidine (CEC, 10-50 microM) inhibited the PE-evoked rise in base line tone, but did not affect the PE-induced facilitation of the neurally evoked contractions nor the facilitation of ACh release. However, CEC increased the area and amplitude of the neurally-evoked contractions by 261+/-33 and 47.2+/-8.4%, respectively. Atropine significantly inhibited the CEC evoked increase in area and amplitude of the electrically evoked contractions (76.5+/-4.8 and 40.8+/-3%, respectively) indicating that CEC facilitated the cholinergic responses of the electrically stimulated bladder strips. It is concluded that alpha(1A) and CEC sensitive alpha(1B) and/or alpha(1D) adrenoceptors are expressed in the rat bladder in different locations. On the cholinergic nerve terminals alpha(1A) adrenoceptors mediate prejunctional facilitation, whereas postjunctional alpha(1B)/alpha(1D) adrenoceptors mediate smooth muscle contraction.     

Effect of chronic cocaine treatment on D2 receptors regulating the release of dopamine and acetylcholine in the nucleus accumbens and striatum.

The inhibition of electrically stimulated [3H]DA and [14C]ACh release by a submaximal concentration of quinpirole was measured 1 week after pretreating rats for 9 days with cocaine (15 mg/kg IP, twice per day). Although this pretreatment significantly enhanced behavioral response to a challenge injection of cocaine when compared with rats pretreated with saline only, no significant differences were apparent in the degree of inhibition of electrically evoked [3H]DA or [14C]ACh release by quinpirole in either the nucleus accumbens or striatum. In addition, the potentiation of electrically evoked [3H]DA release and corresponding inhibition of [14C]ACh release by 10 microM cocaine, measured in striatal slices only, was not significantly different between the two treatment groups. These results suggest that the enhanced behavioral response resulting from chronic cocaine treatment (behavioral sensitization) is not caused by a subsensitivity of D2 terminal autoreceptors or by a supersensitivity of postsynaptic D2 receptors on cholinergic neurons.     

Stimulating effect of methylmercury chloride on [H]acetylcholine release from guinea-pig striatal slices

The effect of methylmercury (MeHg) chloride on the release of [³H]acetylcholine ([³H]ACh) was examined using guinea-pig striatal slices. When the Hg concentration in the striatal slices was less than 10 ppm, MeHg chloride had no effect on spontaneous [³H]ACh release. However, electrically evoked release of [³H]ACh was significantly increased when the tissue Hg concentration was between 0.08 and 3 ppm and was about 160% of the control value at around 0.7 ppm. The increase in evoked ACh release induced by MeHg chloride at low tissue concentrations was partly due to inhibition of the re-uptake of choline. These results suggest that MeHg chloride-induced hyperactivation of cholinergic transmission may be involved in some of the early signs of mercury intoxication.     

Effect of age and COX-2-derived prostanoids on the progression of adult vascular dysfunction in the offspring of diabetic rats

BACKGROUND AND PURPOSE

The present study was designed to determine how diabetes in pregnancy affects vascular function in their offspring, the influence of age and whether COX activation is involved in this effect.

EXPERIMENTAL APPROACH

Relaxation responses to ACh were analysed in mesenteric resistance arteries from the offspring of control rats (O-CR) and those of diabetic rats (O-DR) at 3, 6 and 12 months of age. TxB₂, PGE₂ and PGF_2α release were determined by enzyme immunoassay. COX-1 and COX-2 expression were measured by Western blot analysis.

KEY RESULTS

O-DR developed hypertension from 6 months of age compared with O-CR. In O-DR, relaxation responses to ACh were impaired in all ages studied and were restored by COX-2 inhibition. TP receptor blockade (SQ29548) restored ACh relaxation in arteries from 3-month-old O-DR while TP and EP receptor blockade (SQ29548 + AH6809) was required to restore it in 6-month-old O-DR. In 12-month-old O-DR, ACh relaxation was restored when TP, EP and FP receptors were blocked (SQ29548 + AH6809 + AL8810). ACh-stimulated TxB₂ was higher in all O-DR. ACh-stimulated PGE₂ release was increased in arteries from 6- and 12-month-old O-DR, whereas PGF_2α was increased only in 12-month-old O-DR. COX-2, but not COX-1, expression was higher in O-DR than O-CR.

CONCLUSIONS AND IMPLICATIONS

The results indicate an age-dependent up-regulation of COX-2 coupled to an enhanced formation of vasoconstrictor prostanoids in resistance arteries from O-DR. This effect plays a key role in the pathogenesis of endothelial dysfunction, which in turn could contribute to the progression of vascular dysfunction in these rats.     

Effects of cooling on the levels of acetylcholine, cholinesterase, choline acetyltransferase and the intramural electrical stimulation on the guinea pig ileum

The effects of hypothermia (4 degrees C) on the components of the cholinergic system of the ileal longitudinal muscle and the adherent Auerbach's plexus of the guinea pig ileum have been investigated. Acetylcholine (ACh) content of the muscle was determined by pyrolysis-gas chromatography. It decreased from 119 to a fairly steady level of 16 nmol/g of wet tissue during the first 72 h of cold storage at 4 degrees C under anoxic conditions. Concomitantly, responsiveness to intramural electrical stimulation decreased by 72%. Cholinesterase (ChE) and choline acetyltransferase (ChA) activities each dropped by 40% during this period. However, the de novo synthesis of ACh over the period of study did not change significantly. The response of the preparation to the exogenously applied ACh remained fairly constant suggesting that the changes in the cholinergic receptor are not accountable for the decrease in responsiveness to intramural stimulation. From the results of this study, it has been concluded that cold storage for 5 days leads to: (1) a significant decline in ACh content within 72 h of storage; (2) a decrease in ChE and ChA activities; (3) no significant effect on the cholinergic receptor; and (4) a decrease in responsiveness to intramural electrical stimulation which is probably due to a malfunction of the ACh-releasing mechanism.     

Facilitation of acetylcholine signaling by the dithiocarbamate fungicide propineb.

Dithiocarbamates (DTCs) are used mainly in agriculture as pesticides and as alcohol deterrent drugs. Neurological complications as well as movement disorders characterized by plastic rigidity, muscle twitch and paralysis are the prevailing symptoms in chronically exposed animals and humans. We investigated whether propineb interfered with peripheral cholinergic transmission in various isolated model systems. In electrically stimulated longitudinal muscle-myenteric plexus preparations (LMMPs), propineb (0.01-1000 nM) concentration-dependently enhanced the amplitude of both neurogenic twitch contractions and tritiated acetylcholine ([3H]ACh) release. The maximum percent increase was achieved by 10 nM propineb and was 19% and 14%, respectively. The effect on twitch contractions was partially antagonized by hexamethonium, a ganglionic nicotinic receptor blocker. In unstimulated LMMPs, propineb (10 pM, 10 nM, 10 microM) did not affect contractions to applied acetylcholine (ACh; 1 nM-10 microM), a finding indicating that propineb has no anticholinesterase activity. In human neuroblastoma cells (SH-SY5Y), propineb facilitated ACh release evoked by KCl depolarization. The increase in ACh release was not associated with detectable alterations of intracellular Ca2+([Ca2+]i) homeostasis. Binding studies carried out with alpha-bungarotoxin in striated muscle cells (L6) failed to demonstrate any influence of propineb on both affinity and capacity of skeletal muscle nicotinic receptors. In conclusion, propineb was found to interfere with cholinergic transmission in LMMPs and SH-SY5Y cells. In LMMPs, the potentiation of cholinergic transmission is partly dependent on the activation of ganglionic nicotinic receptors. Other targets relevant to cholinergic transmission seem not to be affected by propineb.     

The toxic effect of an AChE-inhibitor on the cholinergic nervous system in airway smooth muscle

Excessive cholinergic stimulation of presynaptic muscarinic cholinergic receptors, due to complete inhibition of acetylcholinesterase (AChE) by O-(1,2,2-trimethylpropyl)-methyl-phosphonofluoridate (soman), reduced the release of acetylcholine (ACh) from cholinergic nerves in rat bronchi by almost 25%. Furthermore, long-term (40 h) exposure by inhalation of soman (0.45-0.63 mg/m3) reduced the contraction of bronchi induced by ACh by approximately 70%. This is probably due to reduction of the number of muscarinic cholinergic receptors, since there was a reduction in the binding capacity (Bmax) of [3H]QNB by 40%, without any changes in the dissociation constant (Kd).     

Antipsychotic drugs depress acetylcholine release in the Torpedo electric organ, a purely cholinergic system

We used the electric organ of Torpedo, a modified neuromuscular system, to investigate the direct effects of antipsychotic drugs on cholinergic transmission. All the antipsychotic drugs tested inhibited transmission by decreasing the amount of ACh released by nerve impulses. Their potency for this action was as follows: trifluoperazine less than clozapine less than thiethylperazine less than droperidol less than haloperidol less than chlorpromazine = beta-flupentixol less than alpha-flupentixol. Depression of ACh release by antipsychotics was poorly reversible, and was not mediated by dopamine receptors in this system since neither dopamine nor apomorphine had any effect on transmission. Antipsychotics did not act through presynaptic cholinergic receptors since the effect was not antagonized by atropine or quinuclidinyl benzilate. Trifluoperazine had no effect on the total ACh content of the tissue, on the compartmentation of ACh inside and outside synaptic vesicles, or on the rate of ACh turnover or the accumulation of 45Ca observed after repetitive stimulation. We conclude that antipsychotic drugs depress the neurally evoked release of ACh by acting directly on the releasing mechanism.     

Effects of acetylcholinesterase and butyrylcholinesterase inhibition on breathing in mice adapted or not to reduced acetylcholinesterase

We investigated the contributions of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibition to the respiratory dysfunction produced by organophosphates in mice which were adapted or not to low AChE activity. Effects of acute selective inhibition of AChE and BChE on ventilation measured by whole-body plethysmography were compared in mice with either normal AChE activity (wild-type), or mice adapted to a null AChE activity (homozygotes for AChE gene deletion) or adapted to an intermediate level of activity (heterozygotes). In wild-type mice acute reduction of AChE by Huperzine A (1 mg/kg) to the level found in asymptomatic heterozygotes, induced tremors but no respiratory depression, whereas the same dose of Huperzine in heterozygote animals further reduced AChE activity, increased tidal volume (V(T)) and decreased breathing frequency (f(R)). A lethal dose of Huperzine in wild-type mice augmented these respiratory effects, but was ineffective in homozygotes. BChE inhibition by bambuterol was ineffective in wild-type mice and heterozygotes, decreased V(T) in homozygotes adapted to null AChE activity but increased V(T) in wild-type mice acutely treated with Huperzine, also aggravating the cholinergic syndrome. We conclude that: (1) Huperzine does not perturb respiration at a dose inhibiting 40% of AChE, and at a lethal dose does not affect any other enzyme important for respiration; (2) Respiratory function is more sensitive to anticholinesterases in heterozygotes than in wild-type mice; (3) BChE may play distinct roles in respiratory function, because its inhibition has opposite effects on tidal volume depending on whether the mouse has adapted to null AChE or whether AChE has been lowered acutely; (4) BChE inhibition may contribute to the respiratory toxicity of organophosphates.