Acetylcholine suppresses shoot formation and callusing in leaf explants of in vitro raised seedlings of tomato, Lycopersicon esculentum Miller var. Pusa Ruby.

We present experimental evidence to show that acetylcholine (ACh) causes decrease in shoot formation in leaf explants of tomato (Lycopersicon esculentum Miller var Pusa Ruby) when cultured on shoot regeneration medium. The optimum response was obtained at 10(-4) M ACh-enriched medium. ACh also causes decrease in percentage of cultures forming callus and reduces the callus mass. Inhibitors of enzymatic hydrolysis of ACh, neostigmine and physostigmine, also suppresses callogenesis and caulogenesis. On the other hand, the breakdown products of Ach, choline and acetate, do not alter the morphogenic response induced on the shoot regeneration medium. Neostigmine showed optimal reduction in shoot formation at 10(-5) M. The explants cultured on neostigmine augmented medium showed decline in the activity of ACh hydrolyzing enzyme acetylcholinesterase. ACh and neostigmine added together showed marked reduction in callus mass. These results strongly support the role of ACh as a natural regulator of morphogenesis in tomato plants.

Acetylcholinesterase biosensor for carbamate drugs based on tetrathiafulvalene-tetracyanoquinodimethane/ionic liquid conductive gels.

A highly sensitive acetylcholinesterase biosensor was developed for detection of carbamate drugs based on TTF-TCNQ-ionic liquid gel thiocholine sensor. The TTF-TCNQ-ionic/ionic liquid gel was characterized by FT-IR and scanning electron microscopy. The electrocatalytic behavior of TTF-TCNQ-ionic liquid gels toward oxidation of thiocholine was thoroughly investigated. 1-Ethyl-3-methylimidazolium tetracyanoborate gel based sensor allowed amperometric detection of thiocholine at +400 mV vs. Ag/AgCl with a high sensitivity of 55.9±1.2 µA mM(-1)cm(-2) and a low detection limit equal to 7.6 µM. The catalytic rate constant and diffusion constant of thiocholine were estimated from chronoamperometric data. The proposed biosensor based on AChE immobilized in sol-gel matrix was used for the detection of two carbamate therapeutic drugs. Very low detection limits of 26 pM eserine and 0.3 nM neostigmine were achieved. The analysis of spiked tap water proved the biosensor capability to be used as a screening method for detection of carbamate drugs in wastewaters.

Rapid and label-free screening of enzyme inhibitors using segmented flow electrospray ionization mass spectrometry.

Electrospray ionization mass spectrometry (ESI-MS) is an attractive analytical tool for high-throughput screening because of its rapid scan time and ability to detect compounds without need for labels. Impediments to the use of ESI-MS for screening have been the relatively large sample consumed and slow sample introduction rates associated with commonly used flow injection analysis. We have previously shown that by segmenting nanoliter plugs of sample with air, an array of discrete samples can be delivered to a platinum-coated emitter tip for ESI-MS analysis with throughput as high as 0.8 Hz and carry-over between samples less than 0.1%. This method was applied to screening for inhibitors of acetylcholinesterase as a demonstration of the potential of segmented flow ESI-MS for such applications. Each enzyme assay consumed 10 nL of sample. At 1 microL/min infusion rate, 102 samples were analyzed, corresponding to a 0.65 Hz sample analysis rate. Linear quantification of choline was achieved from 200 microM to 10 mM using this method and Z' values were over 0.8 for the assay. Detailed pharmacologic dose-response curves of selected inhibitors were also measured in high-throughput fashion to validate the method.

EphA4 is localized in clathrin-coated and synaptic vesicles in adult mouse brain.

EphA4, a receptor tyrosine kinase, is expressed in various pre-, post- and peri-synaptic organelles and implicated in the regulation of morphological and physiological properties of synapses. It regulates synaptic plasticity by acting as a binding partner for glial ephrin-A3 and possibly other pre- or post-synaptic ephrins. Now, its trafficking mechanisms remain unknown. In this study, we examine the association of EphA4 with transport, clathrin-coated and synaptic vesicles using cell fractionation, vesicle immunoisolation and electron microscopy. EphA4 was found in highly purified fractions of clathrin-coated or synaptic vesicles. It was also detected in vesicles immuno-isolated with antibodies anti-synaptophysin, anti-vesicular glutamate transporter or anti-vesicular GABA transporter; demonstrating its presence in synaptic vesicles. However, it was not detected in immuno-isolated piccolo-bassoon transport vesicles. In vivo and in dissociated cultures, EphA4 was localized by immunoelectron microscopy in vesicular glutamate transporter 1-positive terminals of hippocampal neurons. Remarkably, the cell surface immunofluorescence of EphA4 increased markedly in cultured hippocampal neurons following KCl depolarization. These observations indicate that EphA4 is present in subsets of synaptic vesicles, can be externalized during depolarization, and internalized within clathrin-coated vesicles. This trafficking itinerary may serve to regulate the levels of EphA4 in the synaptic plasma membrane and thereby modulate signaling events that contribute to synaptic plasticity.

Postsynaptic PDLIM5/Enigma Homolog binds SPAR and causes dendritic spine shrinkage.

Dendritic spine morphology is thought to play important roles in synaptic development and plasticity, and morphological derangements in spines are correlated with several neurological disorders. Here, we identified an interaction between Spine-Associated RapGAP (SPAR), a postsynaptic protein that reorganizes actin cytoskeleton and drives dendritic spine head growth, and PDLIM5/Enigma Homolog (ENH), a PDZ-LIM (postsynaptic density-95/Discs large/zona occludens 1-Lin11/Isl-1/Mec3) family member. PDLIM5 has been implicated in susceptibility to bipolar disorder, major depression, and schizophrenia, but its function in neurological disease is poorly understood. We show that PDLIM5 is present in the postsynaptic density, where it promotes decreased dendritic spine head size and longer, filopodia-like morphology. Conversely, RNA interference against PDLIM5 or loss of PDLIM5 interaction with SPAR caused increased spine head diameter. Furthermore, PKC activation promoted delivery of PDLIM5 into dendritic spines and increased its spine colocalization with SPAR. These data reveal new postsynaptic functions for PDLIM5 in shrinkage of dendritic spines that may be relevant to its association with psychiatric illness.

Comparative in vivo effects of parathion on striatal acetylcholine accumulation in adult and aged rats.

Aged rats are more sensitive to the acute toxicity of the prototype organophosphate insecticide, parathion. We compared the acute effects of parathion on diaphragm and brain regional cholinesterase activity, muscarinic receptor binding and striatal acetylcholine levels in 3- and 18-month-old male Sprague-Dawley rats. Adult and aged rats were surgically implanted with a microdialysis cannula into the right striatum 5-7 days prior to parathion treatment. Rats were given either vehicle (peanut oil, 2 ml/kg) or one of a range of dosages of parathion (adult: 1.8, 3.4, 6.0, 9.0, 18 and 27 mg/kg, s.c.; aged: 1.8, 3.4, 6 and 9 mg/kg, s.c.) and body weight, functional signs of toxicity, and nocturnal motor activity were recorded for seven days. Three and seven days after parathion treatment, microdialysis samples were collected and rats were subsequently sacrificed for biochemical measurements. Higher dosages of parathion led to significant time-dependent reductions in body weight in both age groups. Rats in both age groups treated with lower dosages showed few overt signs of cholinergic toxicity while equitoxic high dosages (adult, 27 mg/kg; aged, 9 mg/kg) elicited marked signs of cholinergic toxicity (involuntary movements and SLUD [i.e., acronym for Salivation, Lacrimation, Urination and Defecation] signs) with peak effects being noted 3-4 days after treatment. Nocturnal activity (ambulation and rearing) was reduced in both age groups following parathion dosing, with more prominent effects in adults and rearing being more consistently affected. Dose- and time-dependent inhibition of cholinesterase activity was noted in both diaphragm and striatum. Total muscarinic receptor ([(3)H]quinuclidinyl benzilate, QNB) binding was significantly lower in aged rats, and both total binding and muscarinic agonist ([(3)H]oxotremorine methiodide] binding was significantly reduced in both age-groups treated with the highest dosages of parathion (adult, 27 mg/kg; aged, 9 mg/kg). In contrast to relatively similar levels of cholinesterase inhibition, striatal extracellular acetylcholine levels were significantly lower (2.2- to 2.9-fold) in aged rats at both 3 and 7 day time-points compared to adult rats treated with equitoxic dosages (i.e., 9 and 27 mg/kg, respectively). No age-related differences in in vitro striatal acetylcholine synthesis or in vivo acetylcholine accumulation following direct infusion of the cholinesterase inhibitor neostigmine (1 microM) were noted. While aged rats are more sensitive than adults to the acute toxicity of parathion, lesser acetylcholine accumulation was noted in the striatum of aged rats exhibiting similar levels of cholinesterase inhibition. These findings suggest that lesser acetylcholine accumulation may be required to elicit cholinergic signs in the aged rat, possibly based on aging-associated changes in muscarinic receptor density.

M(2)/M(4)-muscarinic receptors mediate automodulation of acetylcholine outflow from mouse cortex.

Acetylcholine outflow can be modulated through inhibitory presynaptic muscarinic autoreceptors. This study was to identify which subtype is involved in mouse cortex. Five muscarinic antagonists and their ability to elevate stimulation-induced (S-I) acetylcholine outflow were tested in the presence of neostigmine, which decreased S-I outflow. The potency of each antagonist was determined, expressed as a ratio of the potency of each other antagonist and compared with the potency ratios of the antagonists for each of the defined muscarinic receptors (M(1)-M(4)), as recorded in the literature. Linear regression analysis revealed that the data fitted the M(2) (r(2)>0.97) and M(4) (r(2)>0.85) subtypes best, with no correlation for the M(1) and M(3) subtypes.

Neostigmine competitively inhibited nicotinic acetylcholine receptors in sympathetic neurons.

The present experiment investigates the effect of neostigmine on nicotinic acetylcholine receptors (nAChRs) in the cultured neurons from neonatal rat superior cervical ganglia (SCG). Using whole-cell patch clamp techniques, we found that the amplitudes of the currents induced by 50 microM dimethylphenylpiperazinium (DMPP) were 21.5+/-10.7%, 52.9+/-9.2% and 86.9+/-4.9% depressed at the increased concentrations of neostigmine 100, 200 and 400 microM, respectively. The inhibition of neostigmine decreased gradually with the increased concentration of nicotine from 10 to 160 microM. Lineweaver-Burk's double-reversible plot illustrated that neostigmine blocked neuronal nAChRs in a competitive manner. Hyperpolarization of membrane potential from -40 mV to -100 mV did not significantly influence the blockade of neostigmine. Neostigmine could not accelerate the decay of the DMPP-induced currents, neither evoke any detectable currents in SCG neurons. The results indicate that neostigmine depress neuronal nAChRs in a competitive, concentration-dependent and voltage-independent manner, and can not facilitate desensitization of the receptors. The present data suggest that neostigmine blocks neuronal nAChRs by interacting with the ACh binding sites of the receptors.

Studies on the safety of chronically administered intrathecal neostigmine methylsulfate in rats and dogs.

BACKGROUND: The spinal delivery of the cholinesterase inhibitor neostigmine yields analgesia in rats and augments the analgesic effects of alpha 2 agonists in sheep. To assess its activity in humans, preclinical toxicology studies to define its safety were required in two species. METHODS: Rats with chronic intrathecal catheters received daily injections of saline (vehicle) or 5 micrograms/10 microliters or 10 micrograms/10 microliters neostigmine HCl (n = 6/group) for 4 days and were observed for general behavior and nociception (52.5 degrees C hot plate). On day 6, rats were anesthetized and submitted to whole body perfusion/fixation. For dog studies, male beagles were prepared following rigid aseptic precautions with catheters passed from the cisterna magna to the lumbar intrathecal space. Catheters were connected to an external vest-mounted pump. Based on preliminary studies, ten implanted dogs were randomly assigned to receive infusions of neostigmine for 28 days (4 mg/4 ml/day; n = 6) or saline (4 ml/day; n = 4). At 28 days, dogs were anesthetized, cisternal cerebrospinal fluid was obtained, and dogs were submitted to perfusion-fixation. Rat and dog spinal cords were embedded, sectioned, stained, and assessed by the pathologist without knowledge of treatment. RESULTS: In rats, neostigmine produced a dose-dependent increase in hot plate latency, and no tolerance was observed. Mild tremor was observed but was not debilitating. Histopathology revealed a mild fibrotic reaction to the catheter with mixed signs of moderate, acute, and chronic inflammation with no differences between saline or drug groups. In dogs, neostigmine had no effect on blood pressure or on the skin twitch response but produced bradycardia and an increase in muscle tone. At sacrifice, cerebrospinal fluid protein, specific gravity, and glucose were elevated in both saline and neostigmine groups. Histopathology displayed a local reaction to the spinal catheter and a mixed acute and chronic inflammatory reaction. No group differences were observed. These results suggest that, at the neostigmine concentration of 1 mg/ml in the rat and dog and in doses up to 4 mg/day in the dog, there is no evidence of spinal tissue toxicity that can be attributed to the drug. This result, observed in two species, suggests that intrathecal neostigmine given in this manner is without distinguishable toxicity in these two models.

The effect of alkylating agents on the synaptic transmission in the frog's isolated sympathetic ganglion.

The effect of four alkylating agents, such as dibenamine, phenoxybenzamine, mannomustine and cyclophosphamide, was studied on the isolated sympathetic ganglion of the frog. It has been established that all of these agents were able to bind to the nicotinic receptors through the formation of the reactive ethylenimonium ion not only in the frog ganglion but also in the motor end-plate of the rat skeletal muscle. The binding to the ganglionic receptors showed reversible and irreversible phases being dependent not only on the drug concentrations and the incubation time used but the temperature of the medium as well.

Clinical pharmacokinetics of cholinesterase inhibitors.

This review deals mainly with the pharmacokinetics of the reversible quaternary cholinesterase inhibitors neostigmine, pyridostigmine and edrophonium, which are mainly used to antagonise non-depolarising neuromuscular blockade in general anaesthesia and in the symptomatic treatment of myasthenia gravis. Only in the last few years, since the introduction of highly sensitive and selective analytical procedures based on gas and liquid chromatography, have proper pharmacokinetic studies of these drugs become possible. Rapid cooling and addition of internal standard to samples before freezing are important precautions in view of the poor stability of the cholinesterase inhibitors in plasma and blood. Plasma clearances of the reversible quaternary cholinesterase inhibitors are in the range 0.5 to 1.0 L/h/kg and their apparent volumes of distribution range from 0.5 to 1.7 L/kg. Accordingly, the drugs have short plasma elimination half-lives, in the order of 30 to 90 minutes. One to two hours after oral administration of 60 mg pyridostigmine, peak plasma concentrations of 40 to 60 micrograms/L are observed, whereas the plasma concentrations of neostigmine after a 30 mg oral dose are only 1 to 5 micrograms/L. The oral bioavailability of these hydrophilic ionised compounds is low: that of pyridostigmine is approximately 10% and the value for neostigmine is even lower. In spite of the short elimination half-life of pyridostigmine, intraindividual variations in plasma concentration during a dose interval are small in myasthenic patients receiving oral maintenance therapy, probably as a result of slow absorption from the gastrointestinal tract. Severely impaired renal function has been shown to prolong the elimination of neostigmine and pyridostigmine, while methylcellulose has been reported to inhibit the absorption of the latter drug completely. Other pharmacokinetic drug interactions suggested so far do not seem to be of clinical significance. Although a positive correlation has been demonstrated between the plasma concentrations of these drugs and their pharmacological effects as measured by a decrement in muscle response to repetitive nerve stimulation in a single muscle, this relationship is less clear when a global evaluation of muscular function in myasthenia gravis is used. Pharmacokinetic studies of the tertiary reversible cholinesterase inhibitor physostigmine, an important tool in experimental cholinergic neuropharmacology, are still in their initial stages. This drug too is characterised by a short plasma elimination half-life of 20 to 30 minutes.(ABSTRACT TRUNCATED AT 400 WORDS)

Measurement of fasciculations as motor nerve ending discharges in the rat: a dose related effect of neostigmine.

The muscle fasciculations caused by neostigmine and similar agents are the result of a primary drug action on motor nerve endings. Asynchronous, repetitive firing of action potentials are evoked at motor nerve endings which are then transmitted to muscle. A dose-response relationship between neostigmine dose and the rate of/or total neural activity has been established in the rat. This fasciculatory response to neostigmine can serve as an index of motor nerve ending excitability and may be useful in assessing the effects of certain pathological states or drug actions at the neuromuscular junction.

Metabolites of neostigmine and pyridostigmine do not contribute to antagonism of neuromuscular blockade in the dog.

The authors sought to determine whether the metabolites of neostigmine and pyridostigmine contribute to antagonism of neuromuscular blockade. Accordingly, the dose-response relationship, onset and duration of action (n = 60), and pharmacokinetics (n = 22) of neostigmine, pyridostigmine, their metabolites 3-hydroxyphenyltrimethylammonium (PTMA) and 3-hydroxy-N-methylpyridinium (MP), and edrophonium were determined in dogs anesthetized with sodium pentobarbital. The force of contraction of the anterior tibialis muscle was maintained at constant 90% depression by infusing pancuronium. Then, a single iv bolus dose of one of the drugs under study was injected while the pancuronium infusion was continued. Venous blood, urine, and bile were sampled for four hours. Concentrations were determined by liquid chromatographic techniques; a three-compartment pharmacokinetic model was fitted to the serum concentration data. The doses producing 50% antagonism were 6.5, 52, 69, and 40 micrograms/kg for neostigmine, pyridostigmine, edrophonium, and PTMA, respectively. MP was inactive as an antagonist. By comparing approximately equipotent doses, time to peak antagonism (onset) and until 30% of peak antagonism remained (duration) were shorter for both edrophonium and PTMA than for neostigmine and pyridostigmine. Slow distribution and elimination half-lives, volume of distribution at steady state (VDss), and total plasma clearance (Cl) were similar for the drugs except for a smaller Vdss and lower Cl for MP. More than 60% of the dose of each drug was recovered unchanged from urine; less than 1% was recovered from bile. Less than 10% of the dose of neostigmine was recovered as PTMA.(ABSTRACT TRUNCATED AT 250 WORDS)

Clinical pharmacology of edrophonium in infants and children.

The dose-response relationship, onset, duration of action, atropine requirement, and pharmacokinetic variables of edrophonium were determined in infants and children during N2O-halothane anesthesia. The technique of steady state infusion of d-tubocurarine anesthesia. The technique of steady state infusion of d-tubocurarine (dTc) was used to determine the ED50 for edrophonium (i.e., the dose producing 50% antagonism of 90% neuromuscular depression) in 4 infants (145 micrograms/kg) and 12 children (233 microgram/kg). The reported values for ED50 for edrophonium (obtained under similar anesthetic conditions) is 128 micrograms/kg for adults. These three dose-response curves do not differ statistically; however, there was greater variability among infants and children than adults. Time to peak antagonism was similar for all three age groups. Duration of antagonism was determined in six infants and six children and did not differ from the reported value for adults. The optimal dose and time of administration of atropine were established by administering edrophonium (1 mg/kg) and atropine (10-20 micrograms/kg) to 24 infants and children. The smallest changes in heart rate and systolic blood pressure occurred when atropine (10 micrograms/kg) was given 30 s before edrophonium. The pharmacokinetics of edrophonium (1 mg/kg) were studied in four infants and four children and were compared with published values for adults: distribution and elimination half-lives and distribution volumes were similar for the three groups. Total clearance (ml.kg-1.min-1) was greatest for infants (17.8 +/- 1.2) compared with children (14.2 +/- 7.3) and adults (8.3 +/- 2.9). The authors conclude that the dose of edrophonium required toantagonize dTc-induced neuromuscular blockade is similar or possibly greater for infants and children than for adults.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolated parietal cells: [3H]QNB binding to putative cholinergic receptors.

The tritiated muscarinic cholinergic antagonist quinuclidinyl benzilate, [3H]QNB, was used as a direct probe for the detection and characterization of muscarinic cholinergic receptors associated with the particulate fraction of isolated and purified rat gastric muscosal parietal cells. Specific binding is saturable (Bmax = 55 fmol/mg protein, KD = 0.78 nM), shows a single population of binding sites, and has appropriate pharmacological specificity. Nanomolar concentrations of muscarinic cholinergic antagonists, such as atropine and scopolamine, inhibit [3H]QNB binding by 50%, whereas micromolar concentrations are needed for agonists, such as acetylcholine, oxotremorine, and carbamylcholine. Binding is also stereoselective as shown by the more than 1,000-fold difference in inhibitory potencies of the stereoisomers of benzetimide. Noncholinergic agents, including pentagastrin, histamine, and the H2-receptor antagonists cimetidine and metiamide, have little or no effect on [3H]QNB binding at concentrations of 100 microM. These data support the existence of specific parietal cell muscarinic cholinergic receptors with which the secretagogue acetylcholine may directly interact to initiate gastric acid secretion.

Kinetics and metabolism of intramuscular neostigmine in myasthenia gravis.

Neostigmine kinetics and metabolism were studied after intracellular administration to 8 patients with myasthenia gravis. The plasma neostigmine level declined monoexponentially from 21 +/- 2 to 9 +/- 1 ng/ml between 30 and 120 min. The data were interpreted in terms of a 1-compartment model. Estimates of plasma half-life (t1/2) ranged from 51.1 to 90.5 min; apparent volume of distribution varied from 32.0 to 60.6 1; and total body clearance from 434 to 549 ml/min. Approximately 80% of the drug was eliminated in urine within 24 hr either as unchanged neostigmine or its metabolites. Approximately 50% of the dose was eliminated as the unchanged drug, 15% as 3-hydroxyphenyltrimethylammonium, and 15% as other unidentified metabolites. The neostigmine t1/2, based on the urinary excretion of the unchanged drug, ranged from 90.2 to 118.7 min. It was concluded that neostigmine was eliminated by renal and extrarenal mechanisms.