Membrane targeting, shedding and protein interactions of brain acetylcholinesterase

The early stages of Alzheimer's disease are characterized by cholinergic deficits and the preservation of cholinergic function through the use of acetylcholinesterase inhibitors is the basis for current treatments of the disease. Understanding the causes for the loss of basal forebrain cholinergic neurons in neurodegeneration is therefore a key to developing new therapeutics. In this study, we review novel aspects of cholinesterase membrane localization in brain and propose mechanisms for its lipid domain targeting, secretion and protein-protein interactions. In erythrocytes, acetylcholinesterase (AChE) is localized to lipid rafts through a GPI anchor. However, the main splice form of AChE in brain lacks a transmembrane peptide anchor region and is bound to the 'proline-rich membrane anchor', PRiMA, in lipid rafts. Furthermore, AChE is secreted ('shed') from membranes and this shedding is stimulated by cholinergic agonists. Immunocytochemical studies on rat brain have shown that membrane-associated PRiMA immunofluorescence is located selectively at cholinergic neurons of the basal forebrain and striatum. A strong association of AChE with the membrane via PRiMA seems therefore to be a specific requirement of forebrain cholinergic neurons. α7 nicotinic acetylcholine receptors are also associated with lipid rafts where they undergo rapid internalisation on stimulation. We are currently probing the mechanism(s) of AChE shedding, and whether this process and its apparent association with α7 nicotinic acetylcholine receptors and metabolism of the Alzheimer's amyloid precursor protein is determined by its association with lipid raft domains either in normal or pathological situations.     

The lymphocytic cholinergic system and its contribution to the regulation of immune activity

Lymphocytes express most of the cholinergic components found in the nervous system, including acetylcholine (ACh), choline acetyltransferase (ChAT), high affinity choline transporter, muscarinic and nicotinic ACh receptors (mAChRs and nAChRs, respectively), and acetylcholinesterase. Stimulation of T and B cells with ACh or another mAChR agonist elicits intracellular Ca2+ signaling, up-regulation of c-fos expression, increased nitric oxide synthesis and IL-2-induced signal transduction, probably via M3 and M5 mAChR-mediated pathways. Acute stimulation of nAChRs with ACh or nicotine causes rapid and transient Ca2+ signaling in T and B cells, probably via alpha7 nAChR subunit-mediated pathways. Chronic nicotine stimulation, by contrast, down-regulates nAChR expression and suppresses T cell activity. Activation of T cells with phytohemagglutinin or antibodies against cell surface molecules enhances lymphocytic cholinergic transmission by activating expression of ChAT and M5 mAChR, which is suggestive of local cholinergic regulation of immune system activity. This idea is supported by the facts that lymphocytic cholinergic activity reflects well the changes in immune system function seen in animal models of immune deficiency and immune acceleration. Collectively, these data provide a compelling picture in which lymphocytes constitute a cholinergic system that is independent of cholinergic nerves, and which is involved in the regulation of immune function.     

Non-classical actions of cholinesterases: Role in cellular differentiation, tumorigenesis and Alzheimer''s disease

The cholinesterases are members of the serine hydrolase family, which utilizes a serine residue at the active site. Acetylcholinesterase (AChE) is distinguished from butyrylcholinesterase (BChE) by its greater specificity for hydrolysing acetylcholine. The function of AChE at cholinergic synapses is to terminate cholinergic neurotransmission. However, AChE is expressed in tissues that are not directly innervated by cholinergic nerves. AChE and BChE are found in several types of haematopoietic cells. Transient expression of AChE in the brain during embryogenesis suggests that AChE may function in the regulation of neurite outgrowth. Overexpression of cholinesterases has also been correlated with tumorigenesis and abnormal megakaryocytopoiesis. Acetylcholine has been shown to influence cell proliferation and neurite outgrowth through nicotinic and muscarinic receptor-mediated mechanisms and thus, that the expression of AChE and BChE at non-synaptic sites may be associated with a cholinergic function. However, structural homologies between cholinesterases and adhesion proteins indicate that cholinesterases could also function as cell-cell or cell-substrate adhesion molecules. Abnormal expression of AChE and BChE has been detected around the amyloid plaques and neurofibrillary tangles in the brains of patients with Alzheimer's disease. The function of the cholinesterases in these regions of the Alzheimer brain is unknown, but this function is probably unrelated to cholinergic neurotransmission. The presence of abnormal cholinesterase expression in the Alzheimer brain has implications for the pathogenesis of Alzheimer's disease and for therapeutic strategies using cholinesterase inhibitors.     

Actions of a nicotinic agonist,DMPP, on intestinal ion transport invitro

High-dose carbachol (10^?3 M) has previously been shown to cause NaCl absorption in short-circuited rabbit ileum. The mechanism of this effect may be norepinephrine release induced by carbachol activation of presynaptic nicotinic receptors on adrenergic neurons. Norepinephrine then interacts with postsynaptic α-adrenergic receptors on intestinal mucosal cells to stimulate neutral NaCl absorption and inhibit electrogenic bicarbonate secretion. The present paper examines the $\text{in vitro}$ intestinal ion transport effects of DMPP an agent which is more specific than carbachol on nicotinic cholinergic receptors. DMPP (10^?5 M) caused a transient increase followed by prolonged depression of the short-circuit current, increased NaCl absorption and increased tissue conductance. This effect was antagonized by hexamethonium and phentolamine. It is concluded that nicotinic cholinergic agents stimulate norepinephrine release from adrenergic nerves and effect intestinal ion transport just as norepinephrine does.     

The cholinergic anti-inflammatory pathway: a missing link in neuroimmunomodulation

This review outlines the mechanisms underlying the interaction between the nervous and immune systems of the host in response to an immune challenge. The main focus is the cholinergic anti-inflammatory pathway, which we recently described as a novel function of the efferent vagus nerve. This pathway plays a critical role in controlling the inflammatory response through interaction with peripheral a7 subunit-containing nicotinic acetylcholine receptors expressed on macrophages. We describe the modulation of systemic and local inflammation by the cholinergic anti-inflammatory pathway and its function as an interface between the brain and the immune system. The clinical implications of this novel mechanism also are discussed.     

Role of non-neuronal nicotinic acetylcholine receptors in angiogenesis

Angiogenesis is a critical physiological process for cell survival and development. Endothelial cells, necessary for the course of angiogenesis, express several non-neuronal nicotinic acetylcholine receptors (AChRs). The most important functional non-neuronal AChRs are homomeric α7 AChRs and several heteromeric AChRs formed by a combination of α3, α5, β2, and β4 subunits, including α3β4-containing AChRs. In endothelial cells, α7 AChR stimulation indirectly triggers the activation of the integrin αvβ3 receptor and an intracellular MAP kinase (ERK) pathway that mediates angiogenesis. Non-selective cholinergic agonists such as nicotine have been shown to induce angiogenesis, enhancing tumor progression. Moreover, α7 AChR selective antagonists such as α-bungarotoxin and methyllycaconitine as well as the non-specific antagonist mecamylamine have been shown to inhibit endothelial cell proliferation and ultimately blood vessel formation. Exploitation of such pharmacologic properties can lead to the discovery of new specific cholinergic antagonists as anti-cancer therapies. Conversely, the pro-angiogenic effect elicited by specific agonists can be used to treat diseases that respond to revascularization such as diabetic ischemia and atherosclerosis, as well as to accelerate wound healing. In this mini-review we discuss the pharmacological evidence supporting the importance of non-neuronal AChRs in angiogenesis. We also explore potential intracellular mechanisms by which α7 AChR activation mediates this vital cellular process.     

Evidence for operation of nicotinic and muscarinic acetylcholine receptor-dependent survival pathways in human coronary artery endothelial cells

Nicotinic acetylcholine receptors (nAChRs) have recently emerged as critical players in modulation of endothelial function. In particular, studies on endothelial cells from different vascular beds have shown anti-apoptotic actions of nicotinic stimulation, but whether there is actually activation of survival signaling downstream nAChR function has not been explored. In the present work we used human coronary artery endothelial cells (HCAECs) and a pharmacological approach to examine the impact of cholinergic stimulation on survival signaling pathways. Our findings show that cholinergic receptors promote activation of three typical survival routes: the phosphatidyl-inositol-3-kinase (PI3K)/AKT axis, activated downstream muscarinic and nAChRs; the JAK2/STAT3 axis, activated downstream nAChR; and ERK1/2 MAP kinases, activated by both muscarinic acetylcholine receptor (mAChR) and nAChR. Based on their sensitivity to α-bungarotoxin, nicotinic regulation of JAK2/STAT3 and ERK1/2 occurs downstream α7-nAChRs. The present findings suggest that in HCAECs the two cholinergic receptors may act concertedly to induce an efficient survival response of coronary cells when exposed to pro-apoptotic stimuli.     

Nicotine-induced cellular stresses and autophagy in human cancer colon cells: A supportive effect on cell homeostasis via up-regulation of Cox-2 and PGE2 production

Nicotine, one of the active components in cigarette smoke, has been described to contribute to the protective effect of smoking in ulcerative colitis (UC) patients. Furthermore, the nicotinic acetylcholine receptor α7 subunit (α7nAChR) expressed on immune cells, is an essential regulator of inflammation. As intestinal epithelial cells also express α7nAChR, we investigated how nicotine could participate in the homeostasis of intestinal epithelial cells. First, using the human adenocarcinoma cell line HT-29, we revealed that nicotine, which triggers an influx of extracellular Ca²⁺ following α7nAChR stimulation, induces mitochondrial reactive oxygen species (ROS) production associated with a disruption of the mitochondrial membrane potential and endoplasmic reticulum stress. This results in caspase-3 activation, which in turn induces apoptosis. Additionally, we have shown that nicotine induces a PI3-K dependent up-regulation of cyclooxygenase-2 (Cox-2) expression and prostaglandin E2 (PGE₂) production. In this context, we suggest that this key mediator participates in the cytoprotective effects of nicotine against apoptosis by stimulating autophagy in colon cancer cells. Our results provide new insight into one potential mechanism by which nicotine could protect from UC and suggest an anti-inflammatory role for the cholinergic pathway at the epithelial cell level.     

Alpha7 nicotinic acetylcholine receptors in lung inflammation and carcinogenesis: Friends or foes?

The lung tissue expresses the cholinergic system including nicotinic acetylcholine receptors (nAChRs) which included in many physiologic and pathologic processes. Mounting evidence revealed that these receptors have important roles in lung carcinogenesis via modulating either stimulatory or inhibitory signaling pathways. Among different members of nicotinic receptors family, alpha7-subtype of nAChR (α7nAChR) is a critical mediator involved in both inflammatory responses and cancers. Several studies have shown that this receptor is the most powerful regulator of responses that stimulate lung cancer processes such as proliferation, angiogenesis, metastasis, and inhibition of apoptosis. Moreover, aside from its roles in the regulation of cancer pathways, there is growing evidence indicating that α7nAChR has profound impacts on lung inflammation through the cholinergic anti-inflammatory pathway. Regarding such diverse effects as well as the critical roles of nicotine as an activator of α7nAChR on lung cancer pathogenesis, its modulation has emerged as a promising target for drug developments. In this review, we aim to highlight the detrimental as well as the possible beneficial influences of α7nAChR downstream signaling cascades in the control of lung inflammation and cancer-associated properties. Consequently, by considering the significant global burden of lung cancer, delineating the complex influences of α7 receptors would be of great interest in designing novel anticancer and anti-inflammatory strategies for the patients suffering from lung cancer.     

Functional expression of the α7 and α4-containing nicotinic acetylcholine receptors on the neonatal rat carotid body

The carotid bodies (CBs) are chemosensory organs that respond to hypoxemia with transmitter neurosecretion, leading to a respiratory reflex response. It has been proposed that acetylcholine is a key regulator of transmitter release through activation of presynaptic nicotinic acetylcholine receptors (nAChRs). In the present work, we studied the identity of such nAChRs and their contribution to catecholamine release from CBs. Neonatal rat CBs were placed in a recording chamber for electrochemical recordings or disassociated for voltage-clamp studies on isolated cells. Fast nicotine superfusion increases catecholamine release from intact CBs. This response was diminished reversibly by the non-selective nAChR blocker hexamethonium, by the selective α7 blocker α-bungarotoxin and by the α4-containing nAChR blocker erysodine. In isolated CB cells the nAChR agonists nicotine, acetylcholine and cytisine all evoke inward currents with similar potencies. The nicotine-evoked current was fully blocked by mecamylamine and partially inhibited by α-bungarotoxin or erysodine. However, the combination of both α-bungarotoxin an erysodine failed to suppress this response. Immunodetection studies confirm the presence of α7 and α4 subunits in isolated dopaminergic CB cells. Our results show that activation of α7 and/or α4-containing nAChR subtypes have the ability to regulate catecholamine release from intact CB due to activation of fast inward currents expressed in chemoreceptor cells. Therefore, our results suggest that both nAChR subtypes contribute to the cholinergic nicotinic regulation of catecholamine signaling in the carotid body system.     

α7烟碱型乙酰胆碱受体在胆碱能抗炎通路中的作用机制及应用现状

胆碱能抗炎通路是一条神经-免疫调节通路,以中枢神经系统的迷走神经为起点,以神经递质乙酰胆碱为基础,以免疫细胞膜表面的α7烟碱型乙酰胆碱受体(α7 nicotinic acetylcholine receptor, α7 nAChR)为关键作用位点。其中,电信号与化学信号相互转化,激活JAK-STAT3、PI3K-Akt等信号通路,抑制NF-κB的核转位,进而抑制促炎因子的释放,促进抑炎因子的释放,调节和控制机体的炎症,具体的作用机制正在研究中。研究表明,可以通过使用药物激活α7 nAChR和电刺激迷走神经这2种方法,激活胆碱能抗炎通路。其中,激活α7 nAChR具有操作简便、创伤小和效果显著等优势。常用药物有选择性激动剂,例如PNU282987和GTS-21等,非选择性激动剂例如烟碱。多项研究显示,该方法在脓毒症、缺血再灌注、胃肠炎、骨关节炎和自身免疫病等外周器官炎性疾病治疗中都具有一定的效果。α7 nAChR作为胆碱能抗炎通路的关键作用位点,成为许多炎性疾病的潜在治疗靶点,本文对胆碱能抗炎通路中的α7 nAChR所涉及的抗炎机制、通路激活方式,以及其近年来在炎性疾病中的应用进行了综述,为未来研究其具体作用机制和新的治疗靶点提供参考。     

Carrageenans solubilize asymmetric acetylcholinesterase from nicotinic cholinergic synapses

1. Acetylcholinesterase (AChE) catalyzes the hydrolysis of acetylcholine at cholinergic synapses in both vertebrate and invertebrates organisms. 2. The asymmetric synaptic AChE is attached to the extracellular matrix (ECM) of the neuromuscular junction through heparin sulphate proteoglycans (HSPGs). 3. It has been shown previously that heparin-like glycosaminoglycans (GAGs) can solubilize this enzyme from the cholinergic synapses. 4. The present paper describes the solubilization of asymmetric AChE by different marine macroalgal polysaccharides, called carrageenans. 5. Important differences were found among all the carrageenans tested; they released 15-50% of the total AChE activity normally solubilized by heparin. 6. Carrageenans extracted from tetrasporic stages of Iridaea ciliata and I. membranacea were always better extracting agents than those from the cystocarpic stages of these algae, suggesting that lambda-like carrageenans are involved. 7. This hypothesis was confirmed by extracting AChE with purified carrageenans.     

α7-cholinergic receptor mediates vagal induction of splenic norepinephrine

Classically, sympathetic and parasympathetic systems act in opposition to maintain the physiological homeostasis. In this article, we report that both systems work together to restrain systemic inflammation in life-threatening conditions such as sepsis. This study indicates that vagus nerve and cholinergic agonists activate the sympathetic noradrenergic splenic nerve to control systemic inflammation. Unlike adrenalectomy, splenectomy and splenic neurectomy prevent the anti-inflammatory potential of both the vagus nerve and cholinergic agonists, and abrogate their potential to induce splenic and plasma norepinephrine. Splenic nerve stimulation mimics vagal and cholinergic induction of norepinephrine and re-establishes neuromodulation in α7 nicotinic acetylcholine receptor (α7nAChR)-deficient animals. Thus, vagus nerve and cholinergic agonists inhibit systemic inflammation by activating the noradrenergic splenic nerve via the α7nAChR nicotinic receptors. α7nAChR represents a unique molecular link between the parasympathetic and sympathetic system to control inflammation.     

A component of Premarin® enhances multiple cognitive functions and influences nicotinic receptor expression

In women, ovarian hormone loss at menopause has been related to cognitive decline, and some studies suggest that estrogen-containing hormone therapy (HT) can mitigate these effects. Recently, the Women's Health Initiative study found that conjugated equine estrogens, the most commonly prescribed HT, do not benefit cognition. Isolated components of conjugated equine estrogens (tradename Premarin^®) have been evaluated in vitro, with delta^8,9-dehydroestrone (?⁸E1) and equilin showing the strongest neuroprotective profiles. It has not been evaluated whether ?⁸E1 or equilin impact cognition or the cholinergic system, which is affected by other estrogens and known to modulate cognition. Here, in middle-aged, ovariectomized rats, we evaluated the effects of ?⁸E1 and equilin treatments on a cognitive battery and cholinergic nicotinic receptors (nAChR). Specifically, we used ¹²⁵I-labeled epibatidine binding to assay brain nicotinic receptor containing 4α and 2β subunits (α4β2-nAChR), since this nicotinic receptor subtype has been shown previously to be sensitive to other estrogens. ?⁸E1 enhanced spatial working, recent and reference memory. ?⁸E1 also decreased hippocampal and entorhinal cortex α4β2-nAChR expression, which was related to spatial reference memory performance. Equilin treatment did not affect spatial memory or rat α4β2-nAChR expression, and neither estrogen impacted ⁸⁶Rb⁺ efflux, indicating lack of direct action on human α4β2 nAChR function. Both estrogens influenced vaginal smear profiles, uterine weights, and serum luteinizing hormone levels, analogous to classic estrogens. The findings indicate that specific isolated Premarin^® components differ in their ability to affect cognition and nAChR expression. Taken with the works of others showing ?⁸E1-induced benefits on several dimensions of health-related concerns associated with menopause, this body of research identifies ?⁸E1 as a new avenue to be investigated as a potential component of HT that may benefit brain health and function during aging.     

A component of Premarin® enhances multiple cognitive functions and influences nicotinic receptor expression

In women, ovarian hormone loss at menopause has been related to cognitive decline, and some studies suggest that estrogen-containing hormone therapy (HT) can mitigate these effects. Recently, the Women's Health Initiative study found that conjugated equine estrogens, the most commonly prescribed HT, do not benefit cognition. Isolated components of conjugated equine estrogens (tradename Premarin^®) have been evaluated in vitro, with delta^8,9-dehydroestrone (∆⁸E1) and equilin showing the strongest neuroprotective profiles. It has not been evaluated whether ∆⁸E1 or equilin impact cognition or the cholinergic system, which is affected by other estrogens and known to modulate cognition. Here, in middle-aged, ovariectomized rats, we evaluated the effects of ∆⁸E1 and equilin treatments on a cognitive battery and cholinergic nicotinic receptors (nAChR). Specifically, we used ¹²⁵I-labeled epibatidine binding to assay brain nicotinic receptor containing 4α and 2β subunits (α4β2-nAChR), since this nicotinic receptor subtype has been shown previously to be sensitive to other estrogens. ∆⁸E1 enhanced spatial working, recent and reference memory. ∆⁸E1 also decreased hippocampal and entorhinal cortex α4β2-nAChR expression, which was related to spatial reference memory performance. Equilin treatment did not affect spatial memory or rat α4β2-nAChR expression, and neither estrogen impacted ⁸⁶Rb⁺ efflux, indicating lack of direct action on human α4β2 nAChR function. Both estrogens influenced vaginal smear profiles, uterine weights, and serum luteinizing hormone levels, analogous to classic estrogens. The findings indicate that specific isolated Premarin^® components differ in their ability to affect cognition and nAChR expression. Taken with the works of others showing ∆⁸E1-induced benefits on several dimensions of health-related concerns associated with menopause, this body of research identifies ∆⁸E1 as a new avenue to be investigated as a potential component of HT that may benefit brain health and function during aging.     

Capabilities of neurexins in the chick ciliary ganglion

Transcellular interactions between neuroligins (NL) and beta-neurexin have been widely documented to promote maturation and function of both glutamatergic and GABAergic synapses. Recently it has been shown that neuroligin-1 plays a similar role at nicotinic synapses on chick ciliary ganglion neurons in culture, acting from the postsynaptic side to enhance transmitter release from adjacent cholinergic terminals and boost nicotinic input to the cells. We show here that the ciliary ganglion expresses three forms of neuroligin as well as two beta-neurexins and an alpha-neurexin. Overexpression of the beta-neurexins, but not the alpha-neurexin, can induce clustering of endogenous PSD-95 in adjacent neurons, presumably engaging neuroligin in the postsynaptic cell. The trans effects of beta-neurexins are selective; though both alpha3- and alpha7-containing nicotinic receptors are available on opposing cells, beta-neurexins induce coclustering of alpha3- but not alpha7-containing nicotinic receptors. Overexpression of other putative synaptogenic molecules, including SynCAM and L1, are ineffective at trans-clustering of PSD-95 on adjacent neurons. The beta-neurexins also exert a cis effect, coclustering presynaptic markers along with beta-neurexin in neurites juxtaposed to postsynaptic proteins, consistent with organizing presynaptic components as well. Striated muscle, the synaptic target of ciliary neurons in vivo, also expresses neuroligin. The results demonstrate that NL and neurexins are present at multiple sites in nicotinic cholinergic pathways and suggest the possibility of both cis- and trans-interactions to influence nicotinic signaling.     

免疫活性细胞中的非神经元型胆碱能系统

本文对淋巴细胞、巨噬细胞、粒细胞、肥大细胞及胶质细胞等免疫活性细胞中非神经元型胆碱能系统的特点、药理学和病理生理学意义进行了综述。免疫活性细胞,尤其是淋巴细胞中存在完整、独立的胆碱能系统。与神经元型胆碱能系统相比,该系统具有不同特点,如乙酰胆碱(ACh)的合成存在于整个细胞内;ACh可能储存于细胞中某种储存结构,也可能不需储存,而是按需合成并直接释放。免疫活性细胞中的非神经元型胆碱能系统对机体免疫功能及炎症过程可能发挥重要的调控作用。对该系统的深入研究将有助于进一步阐明免疫性疾病、炎症/感染性疾病和神经退行性疾病等的病理生理和发现药物治疗的新靶标。     

In vivo activation of midbrain dopamine neurons via sensitized, high-affinity alpha 6 nicotinic acetylcholine receptors

Alpha6-containing (alpha6*) nicotinic ACh receptors (nAChRs) are selectively expressed in dopamine (DA) neurons and participate in cholinergic transmission. We generated and studied mice with gain-of-function alpha6* nAChRs, which isolate and amplify cholinergic control of DA transmission. In contrast to gene knockouts or pharmacological blockers, which show necessity, we show that activating alpha6* nAChRs and DA neurons is sufficient to cause locomotor hyperactivity. alpha6(L9'S) mice are hyperactive in their home cage and fail to habituate to a novel environment. Selective activation of alpha6* nAChRs with low doses of nicotine, by stimulating DA but not GABA neurons, exaggerates these phenotypes and produces a hyperdopaminergic state in vivo. Experiments with additional nicotinic drugs show that altering agonist efficacy at alpha6* provides fine tuning of DA release and locomotor responses. alpha6*-specific agonists or antagonists may, by targeting endogenous cholinergic mechanisms in midbrain or striatum, provide a method for manipulating DA transmission in neural disorders.     

Cardiac responses of Pacific oyster Crassostrea gigas to agents modulating cholinergic function

To examine the functional effects of cholinergic modulation compounds in oyster hearts and to explore their possible use in monitoring intoxication with acetylcholine-esterase (AChE) inhibitors such as organophosphates, tests were performed with in situ oyster heart preparations. The endogenous cholinergic agonist acetylcholine (ACh), AChE-resistant synthetic agonist carbachol, and the reversible carbamate type of AChE inhibitor physostigmine, all potently depressed spontaneous cardiac contractility. The depression was reversed by extensive washout, or prevented by muscarinic cholinergic antagonist atropine. The irreversible organophosphate type AChE inhibitor parathion or its active metabolite paraoxon at concentrations up to 100 microM failed to depress cardiac contractility. While other reversible AChE inhibitors such neostigmine and pyridostigmine also depressed the contractility, organophosphate AChE inhibitors malathion, diazinon, or phenthoate did not. Despite the differential effect in depressing cardiac function between the reversible and irreversible inhibitors, both of these inhibitors effectively inhibited cardiac AChE activity. The results suggest that the activation of muscarinic cholinergic receptors is coupled to inhibitory cardiac modulation, and organophosphate AChE inhibitors may inhibit only an AChE isozyme located at sites that are not important for control of cardiac activity in oysters.