Effect of diisopropylfluorophosphate on muscarinic and gamma-aminobutyric acid receptors in visual cortex of cats.

Administration of diisopropylfluorophosphate (DFP), an organophosphorus (OP) compound, irreversibly inhibits acetylcholinesterase (AChE) and results in cholinergic hyperactivity. This study investigated muscarinic and gamma-aminobutyric acid (GABA) receptor changes in visual cortex of cats following an acute exposure to DFP. A single acute administration of DFP (4 mg/kg) decreased the number of muscarinic receptors at 2, 10, and 20 hours after treatment. GABA receptors were elevated at 2 and 10 hours but returned to within control levels at 20 hours. No significant alteration in muscarinic or GABA receptor affinity was noted. In all cases cortical AChE activity was inhibited 60-90%. These findings show a down regulation of muscarinic receptors after DFP associated with low AChE activity. GABA receptors also are altered, and may be part of a compensatory mechanism to counteract excess cholinergic stimulation.     

PROPERTIES OF THE EXTERNAL ACETYLCHOLINESTERASE IN GUINEA-PIG IRIS

Abstract— The acetylcholinesterase (AChE) of intact iris, the so-called external AChE, differs in several respects from the AChE in an homogenate of iris, called the total AChE. Maximum enzyme activities of the external and total AChE were obtained with an ACh concentration of 10 and 1.3 m m , respectively. The total AChE exhibited substrate inhibition at high substrate concentrations, whereas the external enzyme did not exhibit substrate inhibition in the range studied. The external AChE activity, when measured at 1.3 m m -ACh. accounted only for 12% of the total enzyme activity. After irreversible inhibition of AChE with diisopropylfluorophosphate (DFP) or methylisocyclopentylfluorophosphate (soman) the external AChE recovered to almost normal values after 48 h, whereas only 30% of the total AChE recovered during this period. Pupillographic studies after inhibition with DFP demonstrated that pupillary diameter had reached normal size after 24 h.
Destruction of the cholinergic input to iris reduced the total AChE activity by 40%, but did not alter the external AChE activity nor its rate of recovery after DFP inhibition. The specific activities of total AChE and total choline acetyltransferase were significantly higher in the sphincter than in the dilator muscle. After such denervation of iris the greatest reduction in total AChE and choline acetyltransferase were found in the sphincter region. After treatment with DFP the total AChE was inhibited to the same extent and recovered at the same rate in both regions.
After extraction of AChE from iris with various salt solutions and detergents, the particulate enzyme recovered faster than the soluble enzyme from DFP inhibition.     

Effect of acute and chronic cholinesterase inhibition with diisopropylfluorophosphate on muscarinic, dopamine, and GABA receptors of the rat striatum

The effects of acute and chronic administration of diisopropylfluorophosphate (DFP) to rats on acetylcholinesterase (AChE) activity (in striatum, medulla, diencephalon, cortex, and medulla) and muscarinic, dopamine (DA), and gamma-aminobutyric acid (GABA) receptor characteristics (in striatum) were investigated. After a single injection of (acute exposure to) DFP, striatal region was found to have the highest degree of AChE inhibition. After daily DFP injections (chronic treatment), all brain regions had the same degree of AChE inhibition, which remained at a steady level despite the regression of the DFP-induced cholinergic overactivity. Acute administration of DFP increased the number of DA and GABA receptors without affecting the muscarinic receptor characteristics. Whereas chronic administration of DFP for either 4 or 14 days reduced the number of muscarinic sites without affecting their affinity, the DFP treatment caused increase in the number of DA and GABA receptors only after 14 days of treatment; however, the increase was considerably lower than that observed after the acute treatment. The in vitro addition of DFP to striatal membranes did not affect DA, GABA, or muscarinic receptors. The results indicate an involvement of GABAergic and dopaminergic systems in the actions of DFP. It is suggested that the GABAergic and dopaminergic involvement may be a part of a compensatory inhibitory process to counteract the excessive cholinergic activity produced by DFP.     

Depression of acetylcholinesterase synthesis following transient cerebral ischemia in rat: pharmacohistochemical and biochemical investigation

The effect of transient cerebral ischemia on acetylcholinesterase (AChE) synthesis was studied in rats by a modified pharmacohistochemical method. The procedure involved in vivo irreversible inhibition of AChE by administration of the inhibitor diisopropyl fluorophosphate (DFP; 1.2 mg/kg b.w., i.m.) 1 h before 30 min forebrain ischemia (the four-vessel occlusion model). At the onset of ischemia, 70-75% of AChE was inhibited in the brain. Recirculation was followed by histochemical and biochemical investigations of newly synthesized AChE in the striatum, septum, cortex and hippocampus. Control sham-operated animals were treated with the same dose of DFP. For correlation, rats not treated with DFP were subjected to the same ischemic procedures and investigated simultaneously. In these rats, significant decrease in AChE activity was found in the striatum, septum and hippocampus during 24 h recirculation. In DFP treated rats, ischemia markedly depressed resynthesis of AChE; after 4 h recirculation, AChE activity was decreased by 45-60% in all investigated areas in comparison with controls and the AChE histochemistry showed only slightly stained neurons in the striatum and septum. Twenty-four hours after ischemia, these neurons were densely stained and the increase in AChE activity indicated a partial recovery of the enzyme synthesis. These results suggest that the depression of AChE synthesis after forebrain ischemia is probably transient, not accompanied by cholinergic neuron degeneration.     

Diisopropylfluorophosphate inhibits acetylcholinesterase activity and disrupts somitogenesis in the zebrafish.

Acetylcholinesterase (AChE) activity, localized histochemically, appeared in the nuclei of presumptive somitic mesodermal cells prior to the onset of somitogenesis. AChE activity appeared in a rostro-caudal sequence, in cells located the equivalent of five somite lengths caudal to the last formed somite. To investigate whether AChE activity was required for somitogenesis, several inhibitors of AChE activity were tested for their ability to block somitogenesis. Diisopropylfluorophosphate (DFP), a broad spectrum inhibitor of serine proteases and related enzymes, was the only AChE inhibitor tested that disrupted somitogenesis. Gastrulae at 50% epiboly exposed continuously to DFP at concentrations between 40 microM and 90 microM completed epiboly, but exhibited a dose-dependent decrease in the number of somites formed, and a parallel decrease in the caudal extent of somite innervation, by 24 hours post-fertilization (h). Fifteen somite (15h) embryos exposed to DFP at the ED50 of 70 microM for 3 hours, followed by recovery to 24h, developed abnormal somites. Approximately five normal somites formed after drug treatment before the first abnormal somite formed. The abnormal somites corresponded in location to that area of the presumptive somitic mesoderm that would have initiated AChE activity while the DFP was present. While exposed to 70 microM DFP, presumptive somites formed and motoneurons extended processes that had initiated AChE activity at the time of treatment with DFP, although at a slower than normal rate. However, embryos exposed to 1 mM DFP for 30 minutes at both the 5 and 15 somite stages, followed by recovery to 24h, developed the normal number of somites but were reduced in the caudal extent of somite innervation, and occasionally developed abnormal primary motoneurons. As with the abnormal somites, the abnormal motoneurons would have initiated AChE activity while the DFP was present. Presumptive somitic mesoderm unable to initiate AChE activity due to inhibition by DFP developed abnormally. While the effects of DFP are not limited to inhibiting AChE, the data support the "clock and wavefront" model proposed for somite formation, and support the hypothesis that AChE activity has a role in somitogenesis in zebrafish.     

Changes in Presynaptic Release of Acetylcholine During Development of Tolerance to the Anticholinesterase, DFP

The rat myenteric plexus was used as a peripheral model for studying muscarinic modulation of acetylcholine (ACh) release from presynaptic muscarinic neurons during development of tolerance to the anticholinesterase agent, diisopropylfluorophosphate (DFP). DFP in arachis oil was administered subcutaneously to intact animals according to both acute and chronic regimens, with arachis oil injections serving as controls. Post-mortem analyses showed that the mean AChE activity level in whole brain was reduced under all DFP conditions to 18.0 +/- 1.4% when compared with the control level. After 10 days of DFP treatment, the AChE level was 22.3 +/- 2.1% of control in the myenteric plexus. There were no significant differences among the treatment groups in resting ACh release. Release evoked by electrical stimulation (difference between stimulated and resting release) in the absence of atropine, i.e., "basal rate," for strips taken at various times after a single injection of DFP did not differ from that for strips from animals receiving arachis oil only. However, basal release for strips from chronically treated subjects was significantly greater than that of controls (p less than 10(-3), although not different from each other. Analysis of variance (ANOVA) for repeated measures showed that there existed a highly significant atropine dependency in strips from all treatments when they were stimulated in concentrations of atropine from 10(-9) to 10(-5) M (p less than 10(-10). Further analyses established that the increases in rates of evoked ACh release as concentrations of atropine increased were similar for strips from chronically treated DFP and arachis oil animals.(ABSTRACT TRUNCATED AT 250 WORDS)     

Central regulation of gastric acetylcholine metabolism and acid output: Analysis using stress and 2-deoxy-d-glucose administration in rats

The stress of immobilization in water caused a significant increase in the activity of choline acetyltransferase (CAT) and acetylcholinesterase (AChE), acetylcholine (ACh) content in the stomach and gastric acid secretion, but a decrease of choline content in rats. The increase in CAT activity began 1 h after the application of stress, peaked in 3 h and gradually decreased to normal within 7 h. Similar alterations in gastric acid secretion were observed. The ACh content in stomach tissue increased 30 min after the application of stress and remained elevated for 2.5 h. The content decreased to control levels after 5 h, and significantly increased again after 7 h. The choline content in stomach tissue significantly decreased 1 and 2 h after stress but returned to normal 3 h after the application. An increase in AChE activity was observed 2 and 7 h after the application of stress but normal levels were found after 4 h. Increases in CAT activity and acid output were also observed following administration of 2-deoxy-d-glucose (2-DG), but no changes in ACh and choline contents or AChE activity were observed. The increases in CAT activity and the acid secretion caused by stress and 2-DG administration were blocked by administration of hexamethonium. These results suggest that increases in gastric CAT, AChE activities and ACh content and a decrease of choline content in the early stages are results of increased vagus nerve activity, which influences gastric acid secretion. Furthermore, they suggest that alterations in ACh content and AChE activity at a later stage are less directly related to the increase in vagus nerve activity.     

Cholinergic hyperinnervation in the cerebral cortex of microencephalic rats does not result in muscarinic receptor down-regulation or in alteration of receptor-stimulated phosphoinositide metabolism

Administration of methylazoxymethanol (MAM; 25 mg/kg) to pregnant rats at gestational day 15 (GD 15) induces a marked reduction of telencephalic areas of the offspring brain. Previous neurochemical studies demonstrated a marked cholinergic hyperinnervation in the cerebral cortex of microencephalic rats. In this study we have evaluated whether this cholinergic hyperinnervation could result in altered functionality of muscarinic receptors. Acetylcholinesterase activity (AChE) was increased by 69% in the cerebral cortex of MAM treated rats confirming a relative hyperinnervation, whereas in the hippocampus and striatum no significant changes were observed. Despite the marked hyperinnervation, in the cerebral cortex of microencephalic rats neither muscarinic receptor-stimulated phosphoinositide metabolism nor muscarinic, receptor density were altered. No differences in receptor density were also observed in the hippocampus and striatum. Chronic diisopropylfluorophosphate (DFP) administration induced a marked decrease of AChE activity and down-regulation of muscarinic receptors whereas atropine administration resulted in receptor up-regulation in cerebral cortex, striatum and hippocampus of both control and MAM rats. The results confirm a relative cholinergic hyperinnervation in the cerebral cortex of microencephalic rats and demonstrate that the regulation of muscarinic receptor-stimulated phosphoinositide metabolism and muscarinic receptor plasticity is not modified in a condition of increased cholinergic presynaptic terminals.     

Occurrence of acetylcholine-hydrolyzing activity at the stele-cortex interface

Acetylcholine (ACh) is a chemical transmitter serving to propagate an electrical perturbation across the synaptic junctions of animals. ACh and AChE have previously been demonstrated to occur in plants. In this work, we detected AChE at the interface between stele and cortex of the mesocotyl of Zea mays by measuring hydrolysis of acetylthiocholine and by liberation of labeled acetate from [1-¹⁴C]ACh. AChE activity was also detected in a crude membrane fraction. The hydrolytic activity is inhibited by neostigmine. Hydrolysis of ACh was also measured after injection of [1-¹⁴C]ACh into kernels of Zea mays and the radioactivity transported into the mesocotyl cortex. A gravity stimulus was then given by placing the plants in a horizontal position. Significantly more radioactivity was found in the lower cortex of horizontally placed seedlings. A working hypothesis is presented for the involvement of ACh and AChE in the tropic response of Z. mays seedlings.     

Sensitivity of brain cholinesterase to cypermethrin toxicity in freshwater teleost Tilapia mossambica

Cypermethrin at sublethal concentrations induced significant changes in acetylcholinesterase (AChE) activity and acetylcholine (ACh) content in the brain tissue of both juvenile and adult-fish. Maximum inhibition of AChE activity is noticed at 6h and 12h after exposure to cypermethrin in juvenile and adult fish respectively. In contrast, the ACh levels registered an elevation in both the cases. During subsequent periods the rate of recovery in AChE activity and ACh content is variable in both the groups.     

The effect of heptyl-physostigmine,a new cholinesterase inhibitor,on the central cholinergic system of the rat

Heptyl-physostigmine (Heptyl-Phy; MF-201) is a new carbamate derivative of physostigmine (Phy) with greater lipophilicity and longer inhibitory action on cholinesterase (ChE) activity than the parent compound. Following single dose administration of 5 mg/kg heptyl-Phy i.m., maximal whole brain acetylcholinesterase (AChE) inhibition (82%) if reached at 60 min. Inhibition of plasma BuChE butyrylcholinesterase (BuChE) remains close to the steady state level (60%) between 120 and 360 min. At 360 min, whole brain AChE activity is still 67% inhibited compared to controls. Inhibition of AChE activity displays brain regional differences which are more significant at 360 min. At this time point, AChe activity in cerebellum is only 40% inhibited while frontal cortex and medial septum are still 80% inhibited. Increases in acetycholine (ACh) levels also show regional differences, however, there is no direct relationship between AChE inhibition and ACh increase. The electrically evoked [³H]ACh release in cortical slices was inhibited only by the highest concentration of heptyl-Phy tested (10^–4M). At this concentration ChE activity was 97% inhibited in vitro. In conclusion, our results demonstrate that heptyl-Phy compares favorably to other reversible cholinesterase inhibitors (ChEI), particularly to Phy as far as producing a more long-lasting inhibition of AChE and a more prolonged increase of ACh in brain with less severe side effects. Therefore, it represents an interesting candidate for cholinomimetic therapy of Alzheimer disease (AD).Dept. of Pharmacology, Shanghai Institute of Materia Medica Chinese Academy of Sciences Shanghai 20031 China.Special issue dedicated to Dr. Paola S. Timiras     

The Muscarinic Receptor Adenylate Cyclase Complex of Rat Striatum: Desensitization Following Chronic Inhibition of Acetylcholinesterase Activity

Chronic inhibition of acetylcholinesterase activity by treatment with diisopropylfluorophosphate (DFP) decreased the capacity of acetylcholine (ACh) acting at a muscarinic receptor to inhibit basal adenylate cyclase activity in homogenates from rat striatum. There was also a loss of the capacity of ACh to inhibit the activation of adenylate cyclase by dopamine. The desensitization of the muscarinic receptor adenylate cyclase complex was associated with a marked attenuation of the capacity of ACh to stimulate a high-affinity GTPase activity present in striatal membranes. The EC50 value of ACh for inhibiting adenylate cyclase and for stimulating GTPase activity increased following treatment with DFP, while the Hill coefficient for both responses was unaltered.     

Effect of local acetylcholinesterase inhibition on sweat rate in humans.

ACh is the neurotransmitter responsible for increasing sweat rate (SR) in humans. Because ACh is rapidly hydrolyzed by acetylcholinesterase (AChE), it is possible that AChE contributes to the modulation of SR. Thus the primary purpose of this project was to identify whether AChE around human sweat glands is capable of modulating SR during local application of various concentrations of ACh in vivo, as well as during a heat stress. In seven subjects, two microdialysis probes were placed in the intradermal space of the forearm. One probe was perfused with the AChE inhibitor neostigmine (10 microM); the adjacent membrane was perfused with the vehicle (Ringer solution). SR over both membranes was monitored via capacitance hygrometry during microdialysis administration of various concentrations of ACh (1 x 10(-7)-2 M) and during whole body heating. SR was significantly greater at the neostigmine-treated site than at the control site during administration of lower concentrations of ACh (1 x 10(-7)-1 x 10(-3) M, P < 0.05), but not during administration of higher concentrations of ACh (1 x 10(-2)-2 M, P > 0.05). Moreover, the core temperature threshold for the onset of sweating at the neostigmine-treated site was significantly reduced relative to that at the control site. However, no differences in SR were observed between sites after 35 min of whole body heating. These results suggest that AChE is capable of modulating SR when ACh concentrations are low to moderate (i.e., when sudomotor activity is low) but is less effective in governing SR after SR has increased substantially.     

大鼠尾壳核衰老变化定量分析：Ⅰ,乙酰胆碱（Ach）系统

应用二异丙基氟磷酸(DFP)控制的乙酰胆碱酯酶(AChE)药物组化染色显示大鼠尾壳核AChE。图像分析系统测定结果表明:老年大鼠(26—27月龄)尾壳核内胆碱能神经元面积、数目及AChE合成速率均较成年大鼠(3—4月龄)减小。这些现象可能是衰老大鼠尾壳核胆碱能神经元功能障碍的生物学基础。     

Two types of asymmetric acetylcholinesterase in chick hindlimb muscle: Developmental profiles,in Vivo and in cell culture,and recovery after inactivation

1. We have analyzed the behavior of two types of asymmetric molecular forms (A forms) of acetylcholinesterase (AChE) during development of chick hindlimb muscle, in vivo and in cell culture, and upon irreversible inactivation of peroneal muscle AChE with diisopropylfluorophosphate (DFP) in vivo. 2. In agreement with previous developmental studies on chick muscle, globular forms of AChE (G forms) are predominant in chick hindlimb at early embryonic ages, being gradually replaced by A forms as hatching (and, therefore, onset of locomotion) approaches. Of the two A-form types, AI appears and accumulates significantly earlier than AII, so that A/G and II/I ratios higher than 1 are attained only at about hatching time. 3. Cultures prepared from 11-day chick embryo hindlimb myoblasts express both types of A forms, with a combined activity of 27% of total AChE after 12 days in culture. AI forms appear again earlier and are much more abundant than type II asymmetric species through the life span of cultures. 4. All AChE activity in the peroneal muscle is irreversibly inactivated by injection of DFP in vivo. The recovery of A forms follows the same sequence described for normal development, with a delayed and slower recovery of AII forms as compared with AI. 5. Several hypotheses involving tail polypeptides or tissue target molecules, or posttranslational interconversion, are proposed to help explain the earlier appearance and accumulation of AI forms in chick muscle.     

Choline Acetyltransferase and Cholinesterases in the Developing Xenopus Retina

To understand the developmental regulation of acetylcholine (ACh) synthesis in the Xenopus retina, the properties of choline acetyltransferase (CAT) and cholinesterase (ChE), as well as histochemical localization of ChE in the retina, were studied during development. CAT activity first became detectable in the developing eyecup at stages 35/36. This was followed by a rapid, 50-fold rise in specific activity between stages 35/36 and 44. Since this rapid rise coincided with an almost identical increase in total ACh synthesis in whole retinae found in previous studies, it is suggested that this increase was sufficient to account for the rapid increase in total ACh synthesis. Moreover, it also correlated with increased rates of synaptogenesis in both the inner and the outer plexiform layers. Total ChE was resolved into specific and nonspecific ChE by the use of tetraisopropylpyrophosphoramide. Total ChE activities first became detectable at stages 35/36. Specific ChE [acetylcholinesterase (AChE)] increased from 50% at stage 39 to 95% of total ChE activities at stage 66. Again, the most rapid increase in both total ChE and AChE activities occurred between stages 35/36 and 44. Histochemical studies showed that AChE was localized predominantly in the two plexiform layers, with the inner plexiform layer more heavily stained at all stages. Moreover, a stratified staining pattern, clearly discerned in the inner plexiform layer, also correlated with synaptogenesis during this early period of retinal development.     

The Na, K-ATPase activity of the separate structural components of the guinea-pig visual system in hypoxia

Different degree of sensitivity to acute hypoxia in vivo has been shown in guinea-pig pigmented epithelium, retina and visual cortex Na, K-ATPase. The highest degree of the enzyme activity inhibition has been noted in the pigmented epithelium (more than 3 times), lower inhibition-in visual cortex (26%) and the lowest-in the retina (18%). In contrast to Na, K-ATPase, hypoxia effect on Mg-ATPase resulted in both inhibition and activation of the enzyme in all 3 structures. Reoxygenation following acute hypoxia increases Na, K-ATPase activity inhibition in the retina and visual cortex. But under reoxygenation the enzyme activity is recovered in the pigmented epithelium. Preliminary administrations of vitamin E completely prevented Na, K-ATPase activity inhibition in all studied structures.     

Choline derivatives and sodium fluoride protect acetylcholinesterase against irreversible inhibition and aging by DFP and paraoxon

A light addressable potentiometric sensor was used to measure acetylcholinesterase (AChE) activity in order to evaluate the protective effects of quaternary compounds and NaF against enzyme phosphorylation and aging by two organophosphates. The use of the immobilized AChE made possible the quick removal of reagents (i.e., organophosphate, 2-pralidoxime, and protectant), thereby permitting accurate determination of AChE activity before and after phosphorylation and aging. Paraoxon was 15-fold more potent in inhibiting AChE than DFP, while the percent aging following phosphorylation by diiso-propylfluorophosphate (DFP) was much higher. Sodium fluoride (NaF), the most effective protectant against phosphorylation and aging, and the quaternary ammonium compounds reduced significantly AChE inhibition by DFP and paraoxon, to similar degrees. Even though the percent AChE activity that was lost to aging was reduced by these agents, aging as a percent of phosphorylated AChE was not reduced. Thus, their major effect was in reducing the percent AChE phosphorylation, which consequently resulted in reduction of total aged AChE. The finding that quaternary ammonium compounds protect against phosphorylation is consonant with the proposed presence of the active site of AChE in an aromatic gorge.     

Acetylcholinesterase activity in visual cortex structures during early visual deprivation

By means of quantitative histochemical methods it has been shown that an early photic deprivation (animals kept in a dark chamber for two months after their birth) leads to a decrease in the activity level of acetylcholinesterase (AChE) in the visual area of the cerebral cortex. With the recovery of the visual function (animals kept in normal photic conditions for two weeks) the AChE activity becomes markedly normalized. The obtained data allow to suggest that the decrease in AChE activity due to deprivation is functionally determined.     

Effect of Chronic Nicotine Treatment on Nicotinic Autoreceptor Function and N-[3H]Methylcarbamylcholine Binding Sites in the Rat Brain

It has been reported that N-methylcarbamylcholine (MCC), a nicotinic agonist, binds to central nicotinic receptors and causes an increase of acetylcholine (ACh) release from certain central cholinergic nerve terminals. The present experiments determine whether these two phenomena change in response to the chronic administration of nicotine, a procedure known to result in an increase in nicotinic binding sites. Chronic nicotine caused a brain region-specific up-regulation of [3H]MCC sites; binding increased in the frontal cortex, parietal cortex, striatum, and hippocampus, but not in the occipital cortex or cerebellum. The effect of nicotine was selective to nicotinic binding sites, because muscarinic sites, both M1 ([ 3H]pirenzepine) and M2 ([3H]ACh), were unaffected by chronic nicotine treatment. MCC increased the release of ACh from the frontal cortex and hippocampus by a calcium-dependent mechanism; MCC did not alter ACh release from striatum or occipital cortex of control animals. The MCC-induced increase in ACh release was not apparent in those animals which had been treated with nicotine. There was a partial recovery of nicotinic autoreceptor function when animals were allowed to recover (4 days) following chronic nicotine treatment, but the density of binding sites remained increased compared to control. Chronic nicotine did not change the potassium-evoked release of ACh from the frontal cortex or hippocampus, but decreased this measure from striatum. It also decreased the ACh content of the striatum, but not that of the cortex or the hippocampus; the activity of choline acetyltransferase was not altered in any of the regions tested.(ABSTRACT TRUNCATED AT 250 WORDS)