Effects of acetylcholinesterase inhibitor paraoxon denote the possibility of non-quantal acetylcholine release in myocardium of different vertebrates

Effects of organophosphorous acetylcholinesterase inhibitor paraoxon were studied in the isolated atrial and ventricular myocardium preparations of a fish (cod), an amphibian (frog) and a mammal (rat) using the microelectrode technique. Incubation of isolated atrium with paraoxon (5 × 10⁻⁶–5 × 10⁻⁵ M) caused significant reduction of action potential duration and marked slowing of sinus rhythm. These effects were abolished by muscarinic blocker atropine and therefore are caused by acetylcholine, which accumulates in the myocardium due to acetylcholinesterase inhibition even in the absence of vagal input. Hemicholinium III is a blocker of high affinity choline-uptake transporters, which are believed to mediate non-quantal release of acetylcholine from cholinergic terminals in different tissues. In the atrial myocardium of all the three studied species, hemicholinium III (10⁻⁵ M) significantly suppressed all the effects of paraoxon. Blocker of parasympathetic ganglionic transmission hexamethonium bromide (10⁻⁴ M) and inhibitor of vesicular acetylcholine transporters vesamicol (10⁻⁵ M) failed to attenuate paraoxon effects. Among ventricular myocardium preparations of three species paraoxon provoked marked cholinergic effects only in frog, hemicholinium III abolished these effects effectively. We conclude that paraoxon stops degradation of acetylcholine in the myocardium and helps to reveal the effects of acetylcholine, which is continuously secreted from the cholinergic nerves in non-quantal manner. Thus, non-quantal release of acetylcholine in the heart is not specific only for mammals, but is also present in the hearts of different vertebrates.     

Early Postdenervation Depolarization Is Controlled by Acetylcholine and Glutamate via Nitric Oxide Regulation of the Chloride Transporter

Resting non-quantal acetylcholine (ACh) and probably glutamate (Glu) release from nerve endings activates M1- and NMDA receptor-mediated Ca²⁺ entry into the sarcoplasm with following activation of NOS and production of NO. This is a trophic message from motoneurons, which keeps the Cl^– transport inactive in the innervated sarcolemma. After denervation, the secretion of ACh and Glu at the neuromuscular junction is eliminated within 3–4 h and the production of NO in the sarcoplasm is lowered. As a result, the Cl^– influx is probably activated by dephosphorylation of the Cl^– transporter with subsequent elevation of intracellular Cl^– concentration. The equilibrium Cl^– potential becomes more positive and the muscle membrane becomes depolarized.     

Mechanisms of acetylcholine-mediated vasodilation in systemic arteries from mourning doves (Zenaida macroura)

For mammals, acetylcholine (ACh) promotes endothelium-dependent vasodilation primarily through nitric oxide (NO) and prostaglandin-mediated pathways, with varying reliance on endothelial-derived hyperpolarizing factors. Currently, no studies have been conducted on small systemic arteries from wild birds. We hypothesized that ACh-mediated vasodilation of isolated small arteries from mourning doves (Zenaida macroura) would likewise depend on endothelial-derived factors. Small resistance mesenteric and cranial tibial (c. tibial) arteries (80–150 μm, inner diameter) were cannulated and pre-constricted to 50 % of resting inner diameter with phenylephrine then exposed to increasing concentrations of ACh (10^?9–10^?5 M) or the NO donor, sodium nitroprusside (SNP; 10^?12–10^?3 M). For mesenteric arteries, ACh-mediated vasodilation was significantly blunted with the potassium channel antagonist tetraethylammonium chloride (TEA, 10 mM); whereas responses were only moderately impaired with endothelial disruption or inhibition of prostaglandins (indomethacin, 10 μM). In contrast, endothelial disruption as well as exposure to TEA largely abolished vasodilatory responses to ACh in c. tibial arteries while no effect of prostaglandin inhibition was observed. For both vascular beds, responses to ACh were moderately dependent on the NO signaling pathway. Inhibition of NO synthase had no impact, despite complete reversal of phenylephrine-mediated tone with SNP, whereas inhibition of soluble guanylate cyclase (sGC) caused minor impairments. Endothelium-independent vasodilation also relied on potassium channels. In summary, ACh-mediated vasodilation of mesenteric and c. tibial arteries occurs through the activation of potassium channels to induce hyperpolarization with moderate reliance on sGC. Prostaglandins likewise play a small role in the vasodilatory response to ACh in mesenteric arteries.     

Muscarinic M1 acetylcholine receptors regulate the non-quantal release of acetylcholine in the rat neuromuscular junction via NO-dependent mechanism

Nitric oxide (NO), previously demonstrated to participate in the regulation of the resting membrane potential in skeletal muscles via muscarinic receptors, also regulates non-quantal acetylcholine (ACh) secretion from rat motor nerve endings. Non-quantal ACh release was estimated by the amplitude of endplate hyperpolarization (H-effect) following a blockade of skeletal muscle post-synaptic nicotinic receptors by (+)-tubocurarine. The muscarinic agonists oxotremorine and muscarine lowered the H-effect and the M1 antagonist pirenzepine prevented this effect occurring at all. Another muscarinic agonist arecaidine but-2-ynyl ester tosylate (ABET), which is more selective for M2 receptors than for M1 receptors and 1,1-dimethyl-4-diphenylacetoxypiperidinium (DAMP), a specific antagonist of M3 cholinergic receptors had no significant effect on the H-effect. The oxotremorine-induced decrease in the H-effect was calcium and calmodulin-dependent. The decrease was negated when either NO synthase was inhibited by N(G)-nitro-L-arginine methyl ester or soluble guanylyl cyclase was inhibited by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one. The target of muscle-derived NO is apparently nerve terminal guanylyl cyclase, because exogenous hemoglobin, acting as an NO scavenger, prevented the oxotremorine-induced drop in the H-effect. These results suggest that oxotremorine (and probably also non-quantal ACh) selectively inhibit the non-quantal secretion of ACh from motor nerve terminals acting on post-synaptic M1 receptors coupled to Ca(2+) channels in the sarcolemma to induce sarcoplasmic Ca(2+)-dependent synthesis and the release of NO. It seems that a substantial part of the H-effect can be physiologically regulated by this negative feedback loop, i.e., by NO from muscle fiber; there is apparently also Ca(2+)- and calmodulin-dependent regulation of ACh non-quantal release in the nerve terminal itself, as calmidazolium inhibition of the calmodulin led to a doubling of the resting H-effect.     

Glutamate regulation of non-quantal release of acetylcholine in the rat neuromuscular junction

Glutamate, previously demonstrated to participate in regulation of the resting membrane potential in skeletal muscles, also regulates non-quantal acetylcholine (ACh) secretion from rat motor nerve endings. Non-quantal ACh secretion was estimated by the amplitude of endplate hyperpolarization (H-effect) following blockade of skeletal muscle post-synaptic nicotinic receptors by (+)-tubocurarine and cholinesterase by armin (diethoxy-p-nitrophenyl phosphate). Glutamate was shown to inhibit non-quantal release but not spontaneous and evoked quantal secretion of ACh. Glutamate-induced decrease of the H-effect was enhanced by glycine. Glycine alone also lowered the H-effect, probably due to potentiation of the effect of endogenous glutamate present in the synaptic cleft. Inhibition of N-methyl-d-aspartate (NMDA) receptors with (+)-5-methyl-10,11-dihydro-5H-dibenzocyclohepten-5,10-imine (MK801), dl-2-amino-5-phosphopentanoic acid (AP5) and 7-chlorokynurenic acid or the elimination of Ca2+ from the bathing solution prevented the glutamate-induced decrease of the H-effect with or without glycine. Inhibition of muscle nitric oxide synthase by NG-nitro-l-arginine methyl ester (l-NAME), soluble guanylyl cyclase by 1H[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) and binding and inactivation of extracellular nitric oxide (NO) by haemoglobin removed the action of glutamate and glycine on the H-effect. The results suggest that glutamate, acting on post-synaptic NMDA receptors to induce sarcoplasmic synthesis and release of NO, selectively inhibits non-quantal secretion of ACh from motor nerve terminals. Non-quantal ACh is known to modulate the resting membrane potential of muscle membrane via control of activity of chloride transport and a decrease in secretion of non-quantal transmitter following muscle denervation triggers the early post-denervation depolarization of muscle fibres.     

Immunocytochemical demonstration of M(1) muscarinic acetylcholine receptors at the presynaptic and postsynaptic membranes of rat diaphragm endplates

M(1)-muscarinic acetylcholine (ACh) receptors (M(1)R) were directly demonstrated immunocytochemically in electronmicroscopic images of rat diaphragm neuromuscular junctions (NMJ). Specific electron-dense granules were located at presynaptic nerve ending membranes and in the sarcolemma in the depths of postsynaptic folds. This first visualization of M(1)R on both sides of the NMJ is in agreement with previous pharmacological data on the regulatory role of M(1)R in quantal and non-quantal ACh release.     

Effect of N-acetylaspartylglutamate (NAAG) on non-quantal and spontaneous quantal release of acetylcholine at the neuromuscular synapse of rat

N-Acetylaspartylglutamate (NAAG), known to be present in rat motor neurons, may participate in neuronal modulation of non-quantal secretion of acetylcholine (ACh) from motor nerve terminals. Non-quantal release of ACh was estimated by the amplitude of the endplate membrane hyperpolarization (H-effect) caused by inhibition of nicotinic receptors by (+)-tubocurarine and acetylcholinesterase by armin (diethoxy-p-nitrophenyl phosphate). Application of exogenous NAAG decreased the H-effect in a dose-dependent manner. The reduction of the H-effect by NAAG was completely removed when N-acetyl-beta-aspartylglutamate (betaNAAG) or 2-(phosphonomethyl)-pentanedioic acid (2-PMPA) was used to inhibit glutamate carboxypeptidase II (GCP II), a presynaptic Schwann cell membrane-associated ectoenzyme that hydrolyzes NAAG to glutamate and N-acetylaspartate. Bath application of glutamate decreased the H-effect similarly to the action of NAAG but N-acetylaspartate was without effect. Inhibition of NMDA receptors by dl-2-amino-5-phosphopentanoic acid, (+)-5-methyl-10,11-dihydro-5H-dibenzocyclohepten-5,10-imine (MK801), and 7-chlorokynurenic acid or inhibition of muscle nitric oxide synthase (NO synthase) by N(G)-nitro-l-arginine methyl ester and 3-bromo-7-nitroindazole completely prevented the decrease of the H-effect by NAAG. These results suggest that glutamate, produced by enzymatic hydrolysis of bath-applied NAAG, can modulate non-quantal secretion of ACh from the presynaptic terminal of the neuromuscular synapse via activation of postsynaptic NMDA receptors and synthesis of nitric oxide (NO) in muscle fibers. NAAG also increased the frequency of miniature endplate potentials (mEPPs) generated by spontaneous quantal secretion of ACh, whereas the mean amplitude and time constants for rise time and for decay of mEPPs did not change.     

Effects of "non-quantal" acetylcholine on postsynaptic membrane senstitivity: Effects of ouabain,and mimicry by exogenous acetylcholine

The development of postsynaptic potentiation (PSP) and desensitization due to "non-quantal" acetylcholine that occurs when acetylcholinesterase (AChE) is inhibited was studied using the Na,K-ATPase inhibitor, ouabain, to alter (initially increasing, then decreasing) the level of non-quantal acetylcholine secretion, and exogenous acetylcholine. When ouabain increased non-quantal secretion the time constant () of the miniature end-plate current (MEPC) decay increased, i.e., PSP developed. The later the application of ouabain relative to inhibition of AChE, the greater the degree of PSP. During the next phase when non-quantal secretion was inhibited the MEPC time course shortened more rapidly than in the controls, i.e., desensitization occurred. If ouabain abolished non-quantal secretion before AChE had been inhibited did not change, and neither PSP nor desensitization developed. When AChE was not inhibited ouabain had no effect on . When ACh was continuously applied at 20 nmol·liter^–1, similar to the nonquantal concentration, the shortening of slowed down, and the signal amplitude declined more rapidly than in controls. Addition of exogenous ACh (50 nmol·liter^–1) after acceleration of MEPC decay had developed caused to increase to its initial value. The combined appearance of PSP and desensitization during the action of non-quantal ACh, and the sustained desensitization after removal of released ACh from the synaptic cleft are discussed.S. V. Kurashova Institute of Medicine, Russian Federation Ministry of Public Health, Kzan. Translated from Neirofiziologiya, Vol. 24, No. 4, pp. 396–404, July–August, 1992.     

The combined effect of Cd2+ and ACh on action potentials of Nitellopsis obtusa cells

Interrelations between the action of acetylcholine (ACh) and cadmium ions (Cd²⁺) on bioelectrogenesis of Nitellopsis obtusa cells were investigated. We analyzed repetitively triggered action potentials (AP), their reproducibility, shape and dynamics of membrane potential after AP induction. ACh significantly increased membrane permeability only at high concentrations (1 mM and 5 mM). Repolarisation level of action potential after the first stimulus was much more positive in all cells treated with ACh as compared to the control. Differences of membrane potentials between points just before the first and the second stimuli were 23.4±.0 mV (control); 40.4±5.9 mV (1 mM ACh solution) and 57.7 ± 8.5 mV (5 mM ACh solution). Cd²⁺ at 20 μM concentration was examined as a possible inhibitor of acetylcholinesterase (AChE) in vivo. We found that cadmium strengthens depolarizing effect of acetylcholine after the first stimulus. The highest velocity of AP repolarization was reduced after ACh application and Cd²⁺strengthened this effect. There were no differences in dynamics of membrane potential after repetitively triggered action potentials in ACh or ACh and Cd²⁺ solutions. This shows that cadmium in small concentration acts as inhibitor of acetylcholinesterase.     

Block by phencyclidine of acetylcholine and barium induced adrenal catecholamine secretion

Activation of the sympathetic system by phencyclidine (PCP) should result in catecholamine release from the adrenals. However, adrenalectomy does not reduce PCP-induced hypertension. In an attempt to rectify this inconsistency, the direct effects of PCP on the bovine adrenal medulla were examined. At (3×10^?6M), PCP reduced the acetylcholine-(ACh)-induced catecholamine release by 50%. Surprisingly, barium-induced secretion of catecholamines was also reduced by PCP. ACh-induced catecholamine release was not altered by 10^?3M 4-aminopyridine (4 AP), the potassium channel blocker. Thus, calcium antagonist actions of PCP and consequent block of catecholamine secretion from adrenal medulla may explain the lack of effect of adrenalectomy on PCP-induced hypertension. Possible contributions of calcium and/or potassium channel blockade to other manifestations of PCP overdosage are discussed.     

Pharmacology of the isolated foregut of the locust Schistocerca gregaria—IV. Characterization of a muscarinic acetylcholine receptor

1. Acetylcholine (ACh; 10⁻⁶ M—7 × 10⁻⁵ M), in the presence of neostigmine (10⁻⁵ M), caused contraction of the locust isolated foregut.2. The effect of ACh was mimicked by carbachol, propionylcholine (PCh), butyrylcholine (BCh), nicotine, SD35651, oxotremorine and muscarine.3. The contractions caused by ACh, BCh and carbachol were abolished by atropine (10⁻⁶M) and reduced by d-tubocurarine (10⁻⁵ M) and decamethonium (5 × 10⁻⁵ M). Hexamethonium and α-bungaro-toxin had no effect on contractions caused by the above agonists.4. None of the antagonists used in this study blocked the contractile effects of nicotine.5. It is concluded that the foregut contains a neuronal nicotinic receptor which, when activated, causes release of ACh which acts on a neuromuscular muscarinic receptor.     

The ionophore X537A (Lasolocid) transiently increases acetylcholine release from rat brain invitro

The effect of X537A on acetylcholine (ACh) release was examined $\text{in vitro}$ in superfused slices of rat cerebrum and striatum. The ionophore (30 μM) induced a transient release of ACh which was not dependent on calcium in the medium. Also in contrast to K⁺-stimulated release, X537A-induced release was not sustained by 10^?5M choline in the superfusion medium and not inhibited by 5 × 10^?4M pentobarbital. The ionophore did not transport ACh or choline from an aqueous to an organic phase. Both K⁺ and X537A inhibited 1 μM (³H) choline uptake into striatal synaptosomes but this effect of X537A was more extensive and less reversible than that caused by K⁺. X537A did not inhibit choline acetyltransferase activity.     

The influence of aminopyridines on Ca2+-dependent evoked release of acetylcholine from rat cortex slices

The release of acetylcholine (ACh) elicited by electrical stimulation was investigated in rat brain cortical slices preloaded with³H-choline. Decreasing the [Ca²⁺]o from 2.5 to 0.3 mM caused a progressive reduction of the evoked release of ACh. 4-Aminopyridine (4AP) or LF14 [(1,1-dimethyl-3-(4-amino-3-pyridyl)], 4×10^–5 M doubled the evoked release of ACh when the [Ca²⁺]o was 2.5 mM and quadrupled it when it was 0.3 mM, to levels higher than those obtained with 2.5 mM [Ca²⁺]o alone. This indicates that both 4AP and LF14 decrease the Ca²⁺ requirements for the evoked release of ACh. The findings of this study indicate that LF14 may be suitable for the symptomatic treatment of senile dementia of Alzheimer's type, presumably caused by dysfuntion of cholinergic transmission in the brain.     

Muscarinic modulation of cardiac activity

The goal of the present review is to report information concerning cardiac innervation or more precisely to approach the modulation of cardiac electrical and mechanical activity by parasympathetic innervation. Acetylcholine (ACh) release by nerve endings from the vagus nerve hyperpolarizes the membrane, shortens action potential (AP) duration and has a negative inotropic effect on cardiac muscle. Toxins are usefull tools in the study of membrane signals. The Caribbean ciguatoxin (C-CTX-1) has a muscarinic effect on frog atrial fibres. The toxin evokes the release of ACh from motoneuron nerve terminals innervating this tissue which allows us to propose a model, similar to the one of the neuromuscular junction (nmj), to describe the events occurring during the triggering and release of ACh. Trachynilysin (TLY) is a proteic toxin which causes an influx of Ca2+ into the cells and releases ACh from nmj synaptic vesicles. TLY has a muscarinic effect on atrial fibres which is explicated in the release of neurotransmitter from the nerve endings generated by the TLY-induced Ca2+ influx. It is known that ACh release from nmj is known to be due to exocytosis of synaptic vesicles via the activation of a proteic complex blocked by botulinum toxins. One of these proteins SNAP-25 is the target of type A botulinum toxin (BoNT/A). The study of hearts isolated from BoNT/A poisoned frogs show that atrial AP is lengthened and reveals the presence of SNAP-25 in nerve endings of this tissue. Moreover, the electrical activity of ventricular muscle is markedly altered; in BoNT/A treated frog, an important outward current activated by internal Ca2+ develops. ACh released from nerve terminals binds to a G protein coupled membrane receptor and activates a K+ channel and other effectors. Five subtypes of muscarinic receptors have been cloned from different tissue (M1, M2, M3, M4) subtypes have been identified in cardiac tissues throughout many species. These receptors coupled with different G-proteins activate different effectors. M1 receptors modulate the cardiac plateau and therefore the magnitude of the peak contraction. M2 receptors are mainly involved in the repolarization phase of the AP and modulate the duration of the peak contraction. The roles of M3 and M4 are not yet clearly defined; however, they may activate K+ currents. In conclusion, ACh releases from parasympathetic nerve endings which innervate cardiac cells follows to similar events (Ca2+ influx; presence of a SNAP-25 protein) to those which produce ACh release from nmj, stimulates different G proteins coupled muscarinic receptors, and activates different effectors involved in the modulation of cardiac electrical and mechanical activity.     

Effects of β-bungarotoxin and taipoxin on contractions of canine airways caused by nerve stimulation

β-Bungarotoxin and taipoxin are snake venoms that have been reported to induce neuromuscular blockade exclusively in somatic motor neurons by a presynaptic mechanism. Taipoxin (1 μg/ml), but not β-bungarotoxin (1 μg/ml), depressed the contractile response of canine airway smooth muscle to electrical stimulation. Taipoxin (1 μg/ml) also slightly depressed the contractile activity of canine airways to two concentrations (5 × 10^?6M and 10^?5M) of exogenously administered acetylcholine. We conclude that taipoxin, but not β-bungarotoxin, induces a weak neuromuscular blockade in the parasympathetic fibers innervating canine airways. The sites of this inhibition are believed to be both presynaptic and postsynaptic.     

Effect of muscarinic agonists on the release of 3H-norepinephrine and 3H-dopamine by potassium and electrical stimulation from rat brain slices

The effect of acetylcholine (ACh) on the release of ³H-norepinephrine (NE) from the cerebellum and ³H-dopamine (DA) from the striatum following the administration of potassium chloride or electrical field stimulation was studied in superfused brain slices. ACh in conc. of 10^?6 and 10^?5M significantly inhibited the release of ³H-NE from cerebellar slices and ³H-DA from striatal slices following 2 min infusion of 50mM potassium chloride. In addition ACh produced a dose-dependent inhibition of the release of ³H-DA from striatal slices following electrical stimulation. The results obtained in the present study are quite consistent with the concept that a muscarinic inhibitory mechanism may be operative on noradrenergic and dopaminergic neurons in the brain.     

Non-quantal secretion of a mediator as factor determining consequences of acetylcholinesterase inhibition]

The mechanism of shortening MEPC decay phase after initial prolongation due to acetylcholinesterase inhibition by armine and neostigmine was studied by use of two-electrode voltage-clamp at the mice diaphragm Factors which switch off non-quantal secretion of acetylcholine from the nerve (acute denervation in vitro, ouabain, high concentration of magnesium ions) only slightly reduced the prolongation of MEPC caused by AChE inhibition. So, postsynaptic potentiation of MEPC by nonquantal ACh is not significant immediately after AChE inhibition. At the same time these factors abolished the process of shortening MEPC decay phase. It is concluded, that desensitization of the postsynaptic membrane induced by nonquantal ACh is the main mechanism of the MEPC shortening and that this mechanism can compensate insufficient AChE activity.     

Acetylcholine potentiation of field stimulated rat vas deferens: A pre-synaptic muscarinic mechanism?

Acetylcholine (ACh) was found to markedly enhance the nerve stimulation induced twitch response of isolated, field-stimulated rat vas deferens (RVD). The ED₂₀₀ (concentration which enhances the twitch response to 200% of control) for this potentiation was 6 × 10^?6M with the maximum twitch response being increased by more than 3 fold (325 ± 30%). Carbachol (ED₂₀₀ = 8.5 × 10^?7M) showed identical results. With each drug the potentiation was competitively antagonized by atropine (10^?7?10^?5M). Physostigmine 10^?8?10^?6M) both enhanced the basal twitch response (215 ± 8% of control at 10^?5M) and the sensitivity of the RVD to ACh (ED₂₀₀ = 3.3 × 10^?7M) but not to carbachol. Atropine, on the other hand reduced the basal twitch response by 18 ± 3% at 10^?5M. Hemicholinium (10^?4M) also reduced the basal twitch responses by 23 ± 5%. ACh (10^?7M?10^?5M) did not modify the responses of unstimulated RVD to norepinephrine or KCl suggesting a pre-synaptic site of action. Taken together these results are compatible with the presence of a pre-junctional, excitatory muscarinic mechanism in the field stimulated RVD. That this cholinergic system may be of physiological significance is supported by the observations that atropine and hemicholinium depress while physostigmine enhances the twitch response in the absence of exogenous ACh.     

Morphine impairs Acetylcholine Release but facilitates Acetylcholine Action at a Skeletal Neuromuscular Junction

THERE is considerable evidence that morphine impairs the release of acetylcholine (ACh) at cholinergic synapses in the brain^1–5, although there are considerable problems in determining the exact site and mechanism of this action. A simple synaptic model would be useful for pursuing this problem and the question arises whether this action of morphine is universal for cholinergic synapses or is restricted to particular sites. Morphine impairs the release of ACh at peripheral muscarinic sites^6–8 but there are no reports about the effects of morphine on ACh release at nicotinic neuromuscular sites. We have reported that both morphine and nalorphine block neuromuscular transmission in amphibian and mammalian skeletal neuromuscular preparations^9,10, apparently as a result of impairment of ACh release. We have now determined by direct measurement that morphine impairs ACh release at a skeletal neuromuscular junction.     

Bisquaternary pyridinium oximes: Comparison of in vitro reactivation potency of compounds bearing aliphatic linkers and heteroaromatic linkers for paraoxon-inhibited electric eel and recombinant human acetylcholinesterase

Oxime reactivators are the drugs of choice for the post-treatment of OP (organophosphorus) intoxication and used widely for mechanistic and kinetic studies of OP-inhibited cholinesterases. The purpose of the present study was to evaluate new oxime compounds to reactivate acetylcholinesterase (AChE) inhibited by the OP paraoxon. Several new bisquaternary pyridinium oximes with heterocyclic linkers along with some known bisquaternary pyridinium oximes bearing aliphatic linkers were synthesized and evaluated for their in vitro reactivation potency against paraoxon-inhibited electric eel acetylcholinesterase (EeAChE) and recombinant human acetylcholinesterase (rHuAChE). Results herein indicate that most of the compounds are better reactivators of EeAChE than of rHuAChE. The reactivation potency of two different classes of compounds with varying linker chains was compared and observed that the structure of the connecting chain is an important factor for the activity of the reactivators. At a higher concentration (10^?3 M), compounds bearing aliphatic linker showed better reactivation than compounds with heterocyclic linkers. Interestingly, oximes with a heterocyclic linker inhibited AChE at higher concentration (10^?3 M), whereas their ability to reactivate was increased at lower concentrations (10^?4 M and 10^?5 M). Compounds bearing either a thiophene linker 26, 46 or a furan linker 31 showed 59%, 49% and 52% reactivation of EeAChE, respectively, at 10^?5 M. These compounds showed 14%, 6% and 15% reactivation of rHuAChE at 10^?4 M. Amongst newly synthesized analogs with heterocyclic linkers (26–35 and 45–46), compound 31, bearing furan linker chain, was found to be the most effective reactivator with a k_r 0.042 min^?1, which is better than obidoxime (3) for paraoxon-inhibited EeAChE. Compound 31 showed a k_r 0.0041 min^?1 that is near equal to pralidoxime (1) for paraoxon-inhibited rHuAChE.