Inhibitor Report for: Neostigmine~Prostigmine

In an era when women were not admitted to the University of Edinburgh and when England's first female physician (Elizabeth Garrett Anderson, 1836-1917) had to venture to Paris, France, to earn her M.D. in 1870, the career of Mary Broadfoot Walker (Figure 1) (1988-1974) stands out for truly remarkable achievement. She is credited with making the most significant discovery in medical therapeutic within the British empire.

This study examined the effects of different levels of acetylcholinesterase (AChE) inhibition on dopaminergic regulation of striatal acetylcholine (ACh) release as estimated by in vivo brain microdialysis. Systemic administration of d-amphetamine (2 or 10 mg/kg) increased the striatal output of ACh when the AChE inhibitor neostigmine (0.1 microM) was present in the perfusion fluid. In contrast, when the same experiments were conducted at 0.01 microM neostigmine, d-amphetamine failed to affect (2 mg/kg) or significantly decreased (10 mg/kg) striatal ACh output. The inhibitory action of the D2 receptor agonist quinpirole (0.2 mg/kg) was significantly greater at 0.01 microM than at 0.1 microM neostigmine. Similarly, there was a nonsignificant trend for the D2 antagonist raclopride (1 mg/kg) to stimulate ACh release to a greater extent at the low neostigmine concentration. In contrast, the stimulant effects of systemic administration of the D1 agonist A-77636 (1.46 mg/kg) on striatal ACh release were the same at the two neostigmine concentrations. These results demonstrate that the concentration of an AChE inhibitor in the perfusion solution can quantitatively and even qualitatively influence the manner in which dopaminergic agents regulate ACh overflow in the striatum. On comparing the present results with earlier reports concerning the effects of d-amphetamine on tissue concentrations of ACh, it is tentatively concluded that a low neostigmine concentration is the more physiologically relevant condition. Under such conditions, at moderate doses d-amphetamine does not appear to alter striatal ACh release, with this likely being due to the opposing actions of D1 and D2 receptors. Nevertheless, until the endogenous interstitial concentrations of striatal ACh can be measured by other methods, the physiological relevance of ACh microdialysis studies in the striatum will remain uncertain.

Intrinsic cholinergic inhibitory pathways present a key modulating system in pain perception. The use of intrathecal (i.t.) acetylcholinesterase-inhibitors, such as neostigmine, result in analgesia in both preclinical and clinical models. However, whether i.t. neostigmine suppresses tonic persistent pain or has peripheral sites of antinociceptive action has not been determined. Thus, we studied central (i.t.) and peripheral (intraarticular; i.a.) neostigmine in a rat inflamed knee joint model. Inhibition of thermal and mechanical hyperalgesia was assessed over 28 h using a modified Hargreaves box and von Frey hairs, respectively. I.t. neostigmine resulted in a dose-dependent thermal analgesia (50% of maximal effective dose [ED50] 0-4 h: 6.6 microg, 24-28 h: 9.4 microg) and mechanical analgesia (ED50 0-4 h: 3.5 microg, 24-28 h: 4.3 microg). I.t. atropine reversed analgesia by i.t. neostigmine. I.a. neostigmine also resulted in an i.a. atropine reversible dose-dependent increase of thermal analgesia, although it did not exceed 60% of a maximal possible analgesic effect with the largest applied dose (ED50 0-4 h: 76.2 microg, 24-28 h: 140.1 microg). Partial suppression of mechanical hyperalgesia was observed after i.a. neostigmine. We conclude that centrally administered neostigmine modulates thermal and mechanical antinociception in this animal model of inflammatory pain. These data suggest a peripheral site of muscarinic antinociception. Implications: This animal study shows that administration of the acetylcholinesterase-inhibitor neostigmine results in enhanced levels of the endogenous neurotransmitter acetylcholine, which seems to act as one of a group of analgesia-modulating compounds at central and peripheral sites in inflammatory pain.

We designed this study to evaluate the postoperative analgesic efficacy and safety of intrathecal (i.t.) neostigmine, i.t. morphine, and their combination in patients undergoing cesarean section under spinal anesthesia. Seventy-nine term parturients were randomly divided into four groups to receive isotonic sodium chloride solution 0.2 mL, neostigmine 25 microg, morphine 100 microg, or the combination of i.t. neostigmine 12.5 microg and morphine 50 microg with i.t. 0.5% hyperbaric bupivacaine 12 mg. There were no significant differences among the four groups with regard to spinal anesthesia, maternal blood pressure and heart rate, or fetal status. Postoperative analgesia was provided by i.v. patient-controlled analgesia (PCA) using fentanyl and ketorolac. Compared with the saline group, the time to first PCA use was significantly longer in the neostigmine group (P < 0.001), with lower 24-h analgesic consumption (P < 0.001). Nausea and vomiting were the most common side effects of i.t. neostigmine (73.7%). Analgesic effectiveness was similar between the neostigmine and morphine groups. Compared with the neostigmine group, the combination group had significantly prolonged analgesic effect and reduced 24-h PCA consumption (P < 0.05) with less severity of nausea and vomiting (P = 0.058). Compared with the morphine group, the combination group tended to have prolonged times to first PCA use (P = 0.054) with a lower incidence of pruritus (P < 0.03). IMPLICATIONS: Intrathecal (i.t.) neostigmine 25 microg produced postoperative analgesia for cesarean section similar to that of i.t. morphine 100 microg, but with a high incidence of nausea and vomiting. The combination of i.t. neostigmine 12.5 microg and i.t. morphine 50 microg may produce better postoperative analgesia with fewer side effects than i.t. neostigmine 25 microg or i.t. morphine 100 microg alone.

The potency of a series of anticholinesterase (anti-ChE) agents and serotonin-related amines as inhibitors of the aryl acylamidase (AAA) activity associated with electric eel acetylcholinesterase (AChE) (EC 3.1.1.7) and horse serum butyrylcholinesterase (BCHE) (EC 3.1.1.8) was examined and compared with the potency of the same compounds as ChE inhibitors. Neostigmine, physostigmine, BW 284C51, (+/-)-huperzine A, E2020, tacrine, edrophonium and heptyl-physostigmine were, in that order, the most potent in inhibiting eel AChE-associated AAA activity, their inhibitor constant (Ki) values being in the range 0.02-0.37 microM. The rank order of the same compounds as AChE inhibitors basically paralleled that of AAA, although they were in general stronger on AChE (Ki = 0.001-0.05). The peripheral anionic site inhibitors propidium and gallamine were inactive on AChE-associated AAA. Serotonin and its derivatives were slightly stronger on AAA (Ki = 7.5-30 microM) than on AChE (Ki = 20-140 microM). Tacrine (IC50 = 0.03 microM), diisopropylfluorophosphate (IC50 = 0.04 microM), heptyl-physostigmine (IC50 = 0.11 microM), physostigmine (IC50 = 0.15 microM) and tetra-iso-propylpyrophosphoramide (iso-OMPA) (IC50 = 0.75 microM) were the most potent in inhibiting horse serum BCHE-associated AAA activity. Serotonin and related amines were very weak on BCHE-associated AAA activity. These results indicate that the inhibitory potencies of the active site anti-ChE agents on the AAA activity associated with eel AChE and horse serum BCHE are closely correlated with their action on the respective ChE. In addition, the efficacy of tacrine, E2020, heptyl-physostigmine and (+/-)-huperzine A in the treatment of Alzheimer's disease is unlikely to be related to the action of these drugs on ChE-associated AAA.

The aim of the present study was to evaluate the effects of neostigmine as a final anaesthetic manoeuvre on colonic anastomoses. A colonic anastomosis was constructed in 40 Sprague-Dawley rats. The animals were divided into two groups: (1) rats receiving intravenous saline solution (placebo); and (2) rats receiving an intravenous injection of neostigmine. The size of the caecum, and the diameters of the pre-anastomotic and post-anastomotic colon were measured during the operation and 4 days after surgery, when all the animals were sacrificed. At this time, the presence of adhesions was also investigated. Each segment containing an anastomosis was removed, and the bursting pressure and bursting wall tension were determined. Loss of caecum diameter was significantly greater in group 2 than in group 1 (P = 0.03). Dilatation and obstruction of the colon were significantly more frequent in group 1 (dilatation, P = 0.01; obstruction, P = 0.047). Also, consumption of water by group 2 was greater than that by group 1 (P = 0.049). No statistically significant differences were found between the diameters of the colon (pre- and post-anastomosis), or with respect to general adhesions and adhesions to the anastomotic line. No significant differences were found between anastomotic resistance (determined in terms of bursting pressure and bursting wall tension) in the two groups. The inclusion of neostigmine in an anaesthetic protocol under experimental setting did not reduce the resistance of colonic anastomoses and did not compromise normal healing. Moreover, obstruction caused by peristaltic weakness might be prevented by the expulsion of stool that is induced by the strong contraction of the colonic smooth muscle.

To investigate whether acetylcholine is released in a similar fashion in different regions of the cortex, in vivo microdialysis was used to measure acetylcholine efflux simultaneously in the medial prefrontal and the frontoparietal cortex, under both basal conditions and following tactile stimulation. Additionally, the effects of including two different concentrations (0.05 microM and 0.5 microM) of a cholinesterase inhibitor (neostigmine) in the perfusion fluid were assessed. Basal levels of acetylcholine (i.e. during non-stimulated sessions) were similar in medial prefrontal and frontoparietal areas. Tactile stimulation reliably increased acetylcholine efflux in a similar fashion (up to 140% increase above baseline) in both cortical areas studied. Predictably, the higher concentration of neostigmine (0.5 microM) increased basal acetylcholine efflux by about 150% from levels observed with the lower neostigmine concentration (0.05 microM), but the concentration of local neostigmine had no effect on either the magnitude or the duration of the increased acetylcholine efflux following tactile stimulation. These results suggest that the pattern of acetylcholine release may be comparable in different areas of the cortex, supporting the idea that cholinergic projections from the basal forebrain to the cortex represent a globally regulated system. Furthermore, while the inclusion of neostigmine in perfusion fluid must be taken into account when interpreting acetylcholine efflux data, it appears that concentrations of up to 0.5 microM do not interfere fundamentally with the lability of cortical acetylcholine efflux in response to behavioural stimulation.

BACKGROUND Clonidine reduces heart rate (HR) responses to atropine, whereas neostigmine causes bradycardia. This study was designed to determine whether clonidine premedication would reduce tachycardia after neostigmine-atropine administration. METHODS: Fifty adult patients without cardiovascular disorders who were scheduled for elective surgeries were randomly assigned to receive approximately 5 microg/kg (oral clonidine clonidine group, n=25) or no clonidine (control group, n=25) 90 min before induction of general anesthesia. After tracheal intubation, anesthesia was maintained with N2O and 12% isoflurane in oxygen while patients were paralyzed with vecuronium and mechanically ventilated. When surgeries were completed, adequate spontaneous respiration, responses to verbal commands, and sustained tetanus by stimulating the ulnar nerve were confirmed, and patients' tracheas were extubated. Then a mixture of 0.05 mg/kg neostigmine and 0.02 mg/kg atropine was administered intravenously over 20 s under stable hemodynamic condition (systolic blood pressure and HR within +/-5% of preceding values), and blood pressure and HR were measured noninvasively at 1-min intervals for 10 min. RESULTS: Increases in HR in the clonidine group were significantly less 1-4 min after neostigmine--atropine injections compared with HR values in the control group. A maximum increase in HR of the clonidine group was also significantly less than the control group (15+/-7 vs. 23+/-10 beats/min; means+/-SD), whereas absolute values of mean blood pressure were similar. Severe bradycardia (HR < 50 beats/min) developed in no patients in either group.
CONCLUSIONS: Premedication with 5 microg/kg oral clonidine attenuates the initial increases in HR without subsequent decreases in HR.

INTRODUCTION: Pain resulting from a usually nonpainful stimulus (allodynia) is a common characteristic of neuropathic pain. Among animal models of allodynia, tight ligature of lumbar spinal nerves has been of special interest because it has been reported to be relieved by sympathectomy. The purpose of this study was to determine whether spinal analgesic agents, which have opposite effects on sympathetic nervous system activity (clonidine decreases it and neostigmine increases it), have differing efficacy in this model. METHODS: Male Sprague-Dawley rats were anesthetized, and the left L5 and L6 spinal nerves were ligated. At least 2 weeks later, a lumbar intrathecal or jugular intravenous catheter was inserted. Withdrawal threshold to mechanical stimulation of the hind paw was determined by application of von Frey filaments before surgery; after surgery; after intrathecal injection of clonidine, neostigmine, or their combination; after intravenous injection of phentolamine or guanethidine; and after surgical sympathectomy. RESULTS: Spinal nerve ligation reduced withdrawal threshold ipsilateral to the lesion. This allodynia was relieved by clonidine (50% block of allodynia at 20+/-1.2 microg and neostigmine (50% block of allodynia at 2+/-0.1 microg, and they interacted synergistically to block allodynia. Neither chemical nor surgical sympathectomy altered allodynia. DISCUSSION: These results disagree with previous observations that mechanical allodynia in this animal model depends on sympathetic nervous system activity. Therefore, intrathecally administered analgesic agents, one that reduces sympathetic outflow from the spinal cord (clonidine) and one that increases it (neostigmine), were similarly effective in this model.

The involvement of muscarinic autoreceptors in the regulation of hippocampal acetylcholine levels during acetylcholinesterase inhibition was examined by perfusing the acetylcholinesterase inhibitor neostigmine bromide (10, 100 or 1000 nM) alone and in the presence of the muscarinic receptor antagonist atropine methylnitrate (10 microM), in resting and behaviourally-activated animals. In resting animals, local perfusion of neostigmine caused a dose-dependent increase in acetylcholine levels. Coadministration of atropine did not affect basal levels in the presence of 10 nM neostigmine, but increased acetylcholine levels approximately four and 11-fold in the presence of 100 nM and 1000 nM neostigmine, respectively. In animals which were behaviourally activated by handling, acetylcholine levels increased two- to three-fold in the presence of all neostigmine concentrations. However, the handling-induced increase in acetylcholine levels was somewhat smaller with 1000 nM neostigmine as compared to 10 nM neostigmine. Atropine had no effect on handling-induced acetylcholine output in the presence of 10 nM neostigmine, but caused greater and longer increases in the presence of 100 nM and 1000 nM neostigmine. These data indicate that acetylcholine levels are greatly reduced by autoinhibition at higher levels of acetylcholine esterase inhibition. The handling-evoked increase in acetylcholine levels is only moderately affected by the level of acetylcholinesterase inhibition, despite the participation of autoreceptors in the handling effect at higher levels of acetylcholinesterase inhibition.

BACKGROUND: Intrathecal injection of local anesthetic agents is associated frequently with hypotension. Conversely, intrathecal administration of neostigmine increases blood pressure by enhancing the accumulation of acetylcholine in the spinal cord. The current study examined directly the interaction of intrathecal injection of bupivacaine and neostigmine on splanchnic sympathetic efferent nerve activity. METHODS: Experiments were performed in rats with intrathecal catheters implanted for the long-term. Rats were anesthetized with ketamine (40 mg/kg, intramuscularly) and alpha-chloralose (60 mg/kg, intraperitoneally). The skin incision sites were infiltrated with 1% lidocaine. Sympathetic efferent activity was recorded from the left greater splanchnic nerve. Sympathetic nerve activity was measured continuously before and after intrathecal injection of saline, 430 nmol (140 microg) of bupivacaine, 25 nmol (7.6 microg) of neostigmine, and a combination of bupivacaine and neostigmine all in volumes of 5 microl. Each group consisted of six animals. RESULTS: Compared with baseline nerve activity, intrathecal injection of neostigmine increased splanchnic sympathetic nerve activity significantly by (mean +/- SEM) 112 +/- 29% after an onset latency of 6.8 +/- 0.9 min. In contrast, bupivacaine decreased splanchnic nerve activity significantly (-65 +/- 13%) after a latency of 3.3 +/- 0.5 min after intrathecal administration. Similar to the effect of saline, intrathecal coadministration of bupivacaine and neostigmine did not alter the splanchnic sympathetic nerve activity significantly. CONCLUSIONS: The current study provides electrophysiologic evidence that intrathecal injection of neostigmine increases whereas bupivacaine decreases sympathetic nerve activity. Further, addition of neostigmine effectively counteracts the inhibitory effect of spinal bupivacaine on the sympathetic nerve activity.

BACKGROUND: Imbalance in cardiac sympathetic tone causes prolongation of the QTc interval of the ECG. On the other hand, impairment of the parasympathetic control of the heart rate caused by anticholinesterase-anticholinergic combinations might also affect the cardiac sympathetic tone and hence the QTc interval of the ECG. The main purpose of the present study was to compare the effects of four anticholinesterase-anticholinergic combinations used for the antagonism of the neuromuscular block on the QTc interval of the ECG, heart rate and arterial pressure. METHODS: Eighty-four ASA class I-II patients with a mean age of 32 to 37 yr undergoing otolaryngological surgery were randomly allocated to one of the following groups: neostigmine 40 microg/kg+glycopyrronium 8 microg/kg (Ne-Glyc), neostigmine 40 microg/kg+atropine 20 microg/kg (Ne-Atr), edrophonium 200 microg/kg+atropine 300 microg (Edr-Atr (1)), edrophonium 500 microg/kg+atropine 7 microg/kg (Edr-Atr (2)). QTc interval and heart rate were measured by a signal processing method based on an IBM/PC/xT-compatible microcomputer and arterial pressure with a sphygmomanometer at 1-min intervals up to 10 min after the injection of the drugs and immediately and 2 min after extubation. The ECG, lead II, was continuously recorded. Neuromuscular block was measured by a Datex relaxograph. RESULTS: In all groups, the most pronounced increase in both QTc interval, heart rate and arterial pressure occurred 1 min after the study drugs and immediately after extubation. In all groups, the mean QTc intervals at 1 and 2 min after the study drugs and after extubation were longer than the upper limit of the normal range (440 ms). Junctional rhythm occurred in 1 to 3 patients in all other groups with the exception of the Edr-Atr(1) group in which no cardiac arrhythmias occurred. At 1 min, the heart rate in the Ne-Atr group was at a significantly higher level than that in the Ne-Glyc group. From 3 to 6 min, the heart rate in the Edr-Atr(2) group and at 3 min in the Edr-Atr(1) group was at a lower level than the heart rate in the Ne-Glyc group. CONCLUSIONS: On the basis of the present results, anticholinesterase-anticholinergic combinations should be avoided in patients having a long QT interval syndrome or a prolonged QT interval from other causes. In addition, the cardiovascular stimulation caused by tracheal extubation should also be avoided in these patients.

1. The recycling process of acetylcholine (ACh) following impulse transmission was studied in terms of muscle potentials evoked by repetitive stimulation in the presence of neostigmine. 2. Wistar rats were anaesthetized with urethane and muscle potentials were recorded with concentric electrodes from their exposed medial gastrocnemius muscles before and after the injection of neostigmine. 3. All potentials before neostigmine treatment were similar in amplitude. A set of 10 stimuli was given at 0.5 Hz 6-8 min after drug administration. The first potential was as large as that before it. The second potential was greatly depressed. Thereafter, potentials gradually recovered. 4. Two sets of 10 stimuli were delivered at a 1 min interval (i.e. with a 40 s rest period between them). The second potential was not depressed so severely in the second set as in the first set. The same procedure was repeated in some rats and the aforementioned phenomenon was noted. When two sets of 10 stimuli were given at an interval of 2 min or more, the second potential was equally depressed in the both sets of stimuli. 5. The recycling of ACh following impulse transmission in the junctional region was revealed in terms of muscle potentials in the presence of neostigmine. This process was activated due to repetitive stimulation. Moreover, the activated state seemed to be maintained for a while after the cessation of stimuli. These results suggest the possible existence of a neural mechanism that can exert an influence on the recycling of ACh following impulse transmission beyond a short period of time.

BACKGROUND: Recently, the spinal administration of neostigmine was shown to produce a dose-dependent analgesia. However, this analgesia is limited by adverse effects. The purpose of this study was to examine the analgesic action of peripheral muscarinic receptors by administering intra-articular neostigmine after operation in patients undergoing knee arthroscopy. METHODS: Sixty patients (classified as American Society of Anesthesiologists status I or II) having arthroscopic meniscus repair during general anesthesia were randomized to receive, in a double-blind manner, after operation 125, 250, or 500 microg intra-articular neostigmine; 2 mg intra-articular morphine; or as control groups intra-articular saline or 500 microg neostigmine given subcutaneously (s.c.). Visual analog pain scores (VAS), duration of analgesia as defined by first demand for patient-controlled analgesia by morphine, and subsequent 48-h consumption of morphine were evaluated. RESULTS: Intra-articular (500 microg) neostigmine resulted in significant VAS reduction 1 h after injection compared with patients given intra-articular saline and with those given intra-articular morphine. Analgesia lasted longer after 500 microg intra-articular neostigmine (350 +/- 126 min) compared with intra-articular morphine (196 +/- 138 min; P < 0.05) or with the control groups (intra-articular saline, 51 +/- 11 min; s.c. neostigmine, 46 +/- 8 min; P < 0.05). The need for supplementary analgesia was significantly higher in control groups than for patients given intra-articular morphine or 500 microg intra-articular neostigmine. No significant analgesic effects were observed for the two lower doses of intra-articular neostigmine. Among all study groups, no adverse effects were observed. CONCLUSIONS: Intra-articular injection of the acetylcholinesterase inhibitor neostigmine produced a moderate but significant analgesic effect. Several mechanisms such as the hyperpolarization of neurons, reduction in the release of pronociceptive neurotransmitters, or activation of the nitric oxide-cyclic guanosine monophosphate pathway might mediate this peripheral cholinergic antinociception by elevating endogenous acetylcholine.

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.

Initial toxicity testing of neostigmine for intrathecal (IT) injection was performed with preservative-free isobaric solution, yet currently available formulations contain the preservatives methyl- and propylparaben and are usually mixed with glucose to yield hyperbaric solutions. Since it has been proposed that preservatives and hyperbaricity increase the risk of neurotoxicity after IT injection, we examined the safety of chronically administered IT neostigmine containing these additives in sheep and rats. Rats receiving daily IT injections of glucose alone or of glucose with preservative-containing neostigmine, 5 and 10 microg, exhibited dose-related antinociception, tremor, and rigidity. In comparison to our previously published study of neostigmine injection in solution without glucose, rats receiving IT neostigmine with glucose displayed less rigidity, tremor, and salivation. Sheep receiving daily injection of glucose alone or with preservative-containing neostigmine, 1 mg, for 14 days exhibited no histologic evidence of neurotoxicity, nor did they exhibit abnormalities in cerebrospinal fluid chemistry aside from those caused by inflammation. Spinal cord histologic examination in both species revealed fibrosis and inflammation secondary to the catheter without evidence of neuronal damage. These studies support the safety of paraben- and glucose-containing IT neostigmine.

In an era when women were not admitted to the University of Edinburgh and when England's first female physician (Elizabeth Garrett Anderson, 1836-1917) had to venture to Paris, France, to earn her M.D. in 1870, the career of Mary Broadfoot Walker (Figure 1) (1988-1974) stands out for truly remarkable achievement. She is credited with making the most significant discovery in medical therapeutic within the British empire.

A clinical trial was conducted to evaluate the postoperative analgesic efficacy and the safety of intrathecal neostigmine in patients undergoing anterior and posterior vaginoplasty under spinal anaesthesia. Thirty-six patients were randomly divided into three groups to receive: normal saline (1 ml), morphine (100 micrograms in 1 ml of saline) or neostigmine (100 micrograms in 1 ml of saline) intrathecally just before a spinal injection of hyperbaric bupivacaine (0.5%, 4 ml). The mean [SD] time to the first analgesic (nonsteroidal anti-inflammatory drug) administration was significantly prolonged by intrathecal neostigmine (10.7 [4.3] h) and morphine (15.3 [3.0] h) compared with saline (4.5 [1.0] h). The three groups also differed in the number of patients requiring subcutaneous morphine to complement the analgesia provided by the intramuscular nonsteroidal anti-inflammatory drugs and the mean [SD] times for their administration: eight patients in the saline group (8.0 [3.8] h), one patient in the morphine group (18 h) and two patients in the neostigmine group (8 and 12.9 h). The morphine and neostigmine groups showed similar analgesic effectiveness. The characteristics of spinal anaesthesia were not modified by intrathecal morphine or neostigmine. Severe nausea and vomiting, sweating and distress during surgery were the most obvious adverse effects of intrathecal neostigmine. On the other hand, less hypotension was observed in the neostigmine group. The usefulness of intrathecal neostigmine as the sole postoperative analgesic may be restricted by the severity of its adverse effects.

Intrathecal (IT) neostigmine produces analgesia in animals and humans and enhances systemic opioid analgesia. To examine the safety of IT neostigmine for eventual use in obstetrics, we studied 24 healthy, term pregnant patients scheduled to receive elective cesarean section using a combined spinal-epidural anesthetic. Using an open-label, dose-ranging design, patients received either IT placebo or neostigmine 10, 30, or 100 microg in a 1-mL solution of 5% glucose in normal saline followed in 15 min by 2% epidural lidocaine for cesarean section. Neostigmine did not affect fetal heart rate tracings or Apgar scores. The women received patient-controlled analgesia intravenous morphine postoperatively. Compared with the glucose control, neostigmine produced a dose-independent reduction in postoperative morphine use. Cumulative average 24-h morphine use was 82 +/- 7 mg for women receiving IT placebo and 50 +/- 8 mg for women receiving IT neostigmine (P < 0.003). Hourly morphine use was significantly reduced in the neostigmine groups for 10 h postoperatively. These data indicate that IT neostigmine can produce 10 h of post-cesarean section analgesia without adverse fetal effects and support cautious further prospective study.

We examined the influence of the concentration of sevoflurane and the degree of muscle block at the time of reversal on the activity of neostigmine. Ninety ASA 1-2 patients were anaesthetised with 0.2, 0.7 or 1.2 MAC of sevoflurane (30 patients each) in 66% nitrous oxide in oxygen. The electromyographic (EMG) response of the adductor digiti minimi was monitored at 20-s intervals after train-of-four stimulation of the ulnar nerve. The initial neuromuscular block was produced by vecuronium 100 micrograms.kg-1. When the amplitude of the first response (T1) values had recovered to 10%, 25% or 40% of the control, neostigmine 40 micrograms.kg-1 was administered. The train-of-four ratio values were recorded at 1-min intervals during the subsequent 15-min period. Higher endtidal concentrations (p < 0.0001) and more pronounced block at the time of reversal (p < 0.0001) were associated with a delayed recovery in the train-of-four ratio. In addition, the train-of-four ratio 15 min after neostigmine administration was more dependent on the sevoflurane concentration than on the degree of block present (p < 0.0001). These results confirm that neostigmine (40 micrograms.kg-1) can reverse vecuronium-induced but not sevoflurane-induced neuromuscular block.

The analgesic efficacy of cholinergic agonists and anticholinesterase agents has been widely recognized. The analgesic effect obtained by activating cholinergic mechanisms, however, seems to depend on the experimental pain model utilized for its evaluation. The antinociceptive effect of intraspinal neostigmine was examined in rats submitted concurrently to the tail flick and formalin tests. Neostigmine (8.25 and 16.5 nmol) produced a dose-dependent antinociceptive effect in the tail flick test (a model of phasic pain) and reduced the first phase (phasic pain) of the animal response to formalin also in a dose-dependent manner. The second phase (tonic pain) of the response to formalin, however, was slightly reduced after a longer period of time only by the higher dose of the anticholinesterase. The effect of neostigmine was not significantly different when the drug was injected into rats submitted exclusively to the tail flick test. The second phase of the animal response to formalin was slightly reduced by neostigmine (8.25 nmol) and strongly inhibited by the higher dose of the anticholinesterase when injection was made after the first phase. We conclude that phasic and tonic pain can both be controlled by high doses of neostigmine. In addition, we show that inhibition by a lower dose of neostigmine of the formalin-induced phasic pain did not prevent the subsequent occurrence of tonic pain produced by the irritant.

Sinus tachycardia caused by circulating catecholamines in the setting of congestive heart failure may impair systemic perfusion because of decreased diastolic filling time. We report the case of a patient with Wolff-Parkinson-White syndrome with angina and cardiogenic shock who improved dramatically following administration of neostigmine. Cardiac output, blood pressure, and stroke volume increased as heart rate was reduced. A previous attempt at heart rate control, in the same patient, using a low dose beta-antagonist, precipitated hemodynamic collapse. The remarkable recovery of our patient suggests that acetylcholinesterase inhibitors may warrant further investigation in patients with severe sinus tachycardia.

OBJECTIVE In this study the effect of glyceryl trinitrate on the prostigmine-morphine-induced sphincter of Oddi spasm was evaluated in nine female patients with sphincter of Oddi dyskinesia. METHOD: Sphincter of Oddi spasm was induced by prostigmine-morphine administration (0.5 mg prostigmine intramuscularly and 10 mg morphine subcutaneously) and visualized by quantitative hepatobiliary scintigraphy. The entire procedure was repeated during glyceryl trinitrate infusion (Nitrolingual 1 microg/kg/min for 120 min). RESULTS: Prostigmine-morphine provocation caused significant increases in the time to peak activity (Tmax) over the hepatic hilum (HH: 34.33 +/- 5.05 vs. 22.77 +/- 3.26) and the common bile duct (CBD: 60.44 +/- 5.99 vs. 40.0 +/- 2.88) and in the half-time of excretion (T1/2) over the liver parenchyma (LP: 120.04 +/- 16.01 vs. 27.37 +/- 2.19), HH (117.61 +/- 14.71 vs. 31.85 +/- 3.99) and CBD (158.11 +/- 9.18 vs. 40.1 +/- 6.24), indicating a complete spasm at the level of the sphincter of Oddi. Glyceryl trinitrate infusion completely normalized the prostigmine-morphine-induced alterations in these quantitative parameters (TmaX over the LP: 11.33 +/- 1.13; over the HH: 18.88 +/- 1.48; and over the CBD: 36.22 +/- 1.92; and T1/2 over the LP: 28.21 +/- 1.83; over the HH: 33.42 +/- 3.10; and over the CBD: 41.66 +/- 6.33), suggesting an effective sphincter-relaxing effect of glyceryl trinitrate. CONCLUSION: These results provide the first evidence of the effectiveness of glyceryl trinitrate on the morphine-induced sphincter of Oddi spasm in humans. Since glyceryl trinitrate is able to overcome even the drastic effect of morphine, it might be of relevance in the treatment of sphincter of Oddi dyskinesia.

1. Human isolated pulmonary vessels were treated with cholinesterase (ChE) inhibitors to determine the role of these enzymes in regulating vascular muscle tone. In addition, kinetic parameters were determined for acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) in human pulmonary vessel homogenates. 2. Carbachol (CCh) and acetylcholine (ACh) were equipotent contractile agonists in human pulmonary arteries (pD2 values, 5.28 +/- 0.05 and 5.65 +/- 0.16; Emax, 0.91 +/- 0.26 and 0.98 +/- 0.30 g wt. for CCh and ACh, respectively; n = 7). In venous preparations, ACh was ineffective and CCh induced small contractions (Emax, 0.08 +/- 0.04 g wt; n = 13). 3. In human pulmonary arteries following pretreatment with tetraisopropylpyrophosphoramide (iso-OMPA, 100 microM), an increased sensitivity to the contractile agonist ACh was observed (pD2 values, 5.80 +/- 0.13 and 6.37 +/- 0.19 for control and treated preparations, respectively; n = 5). This pretreatment had no effect on the CCh concentration response curve. In contrast, human pulmonary veins pretreated with iso-OMPA failed to elicit a contractile response to ACh. 4. Neither Iso-OMPA nor neostigmine elicited concentration-dependent contractions in human isolated pulmonary arteries or veins. These results suggest the absence of sufficient spontaneous release of ACh to modulate human pulmonary vessel basal tone. 5. CCh was less potent than ACh in relaxing precontracted human isolated pulmonary arteries (pD2 value, CCh: 6.55 +/- 0.15 and ACh: 7.16 +/- 0.13, n = 4) and veins (pD2 value, CCh: 4.95 +/- 0.13; n = 5 and ACh: 5.56 +/- 0.17; n = 6). Pretreatment of vessels with either iso-OMPA or neostigmine did not modify ACh relaxant responses in either type of preparation. 6. In human pulmonary veins, the ChE activity was two fold greater than in arteries (n = 6). Vmax for AChE was 1.73 +/- 0.24 and 3.36 +/- 0.26 miu mg-1 protein in arteries and veins, respectively, whereas Vss for BChE was 1.83 +/- 0.22 and 4.71 +/- 0.17 miu mg-1 protein, in these respectively. 7. In human pulmonary arteries, BChE activity may play a role in the smooth muscle contraction but not on the smooth muscle endothelium-dependent relaxation induced by ACh. A role for ChE activity in the control of venous tone is presently difficult to observe, even though this tissue contains a greater amount of enzyme than the artery.

PURPOSE: This study evaluated the effect of neostigmine on heart rate in cardiac transplant patients. METHODS: Neostigmine (2.5-50 micrograms.kg-1) was administered to ASA 1 or 2 patients with normally innervated hearts (controls), and to patients who had undergone recent (< six months before study) or remote (> six months before study) cardiac transplantation. RESULTS: Baseline heart rate was 66 +/- 3 beats.min-1 in controls (n = 10, mean +/- SEM), which was slower than that observed in recently (95 +/- 4 beats.min-1, n = 15, P < 0.001) and in remotely (88 +/- 3 beats.min-1, n = 16, P < 0.001) transplanted patients. Neostigmine produced a dose-dependent decrease in heart rate in all patients. Controls were the most sensitive to neostigmine, with a 10% decrease in heart rate produced by an estimated dose of 5.0 +/- 1.0 micrograms.kg-1. The recently transplanted group was the least sensitive, with the maximum dose producing only an 8.3 +/- 0.9% reduction. The response to neostigmine of the remotely transplanted patients was variable. The estimated dose to produce a 10% decrease in heart rate in this group was 24 +/- 6 micrograms.kg-1 which was greater than that for controls (P = 0.008). Administration of atropine (1.2 mg) reversed the neostigmine-induced bradycardia in all three groups. Reversal of the bradycardia consisted of a transient peak increase in heart rate in controls to 145 +/- 6% of baseline, a value which was greater than that observed in recent (103 +/- 1%, P < 0.001) and in remote (109 +/- 3%, P < 0.001) transplants. CONCLUSIONS: Neostigmine produces a dose-dependent bradycardia in heart transplant patients. Some remotely transplanted patients may be particularly sensitive to the bradycardic effects of neostigmine.

PURPOSE: This report describes the effects of neostigmine on heart rate in the same patient following recent and remote cardiac transplantation. CLINICAL FEATURES: Eighty-six months following the first transplant, neostigmine 5.0 micrograms.kg-1 i.v. produced a 10% reduction in heart rate which was reversed by atropine 1.2 mg. For 24 months prior to this initial study, the patient experienced angina, suggesting cardiac afferent reinnervation. Three months after the second heart transplant, a second study showed that a six-fold increase in the dose of neostigmine, 30.0 micrograms.kg-1, only produced a 3.5% reduction in heart rate which was reversed by atropine 1.2 mg. CONCLUSIONS: These observations indicate that neostigmine produces bradycardia following cardiac transplantation, and suggest that a greater response may be observed in remotely than in recently transplanted patients.

The bradycardia produced by neostigmine and edrophonium was examined according to its relation to cholinesterase inhibition and to its sensitivity to block by muscarinic receptor antagonists. For comparison, the ability of muscarinic antagonists to block the bradycardia produced by electrical stimulation of the vagus nerve was determined. METHODS: Cats were anaesthetized, vagotomized and propranolol-treated. Heart rate was continuously recorded. Erythrocyte cholinesterase activity of arterial blood was measured using a radiometric technique. The right vagus nerve was isolated for electrical stimulation. The muscarinic antagonists used were atropine, glycopyrrolate, pancuronium, gallamine, and AFDX-116. RESULTS: Neostigmine produced a dose-dependent decrease in cholinesterase activity which reached a plateau at a cumulative dose of 0.16 mg.kg-1 (ED50 0.009 +/- 0.003 mg.kg-1). Neostigmine produced a dose-dependent decrease in heart rate with the dose-response relationship (ED50 0.1 +/- 0.01 mg.kg-1; P = 0.0006) shifted to the right of that for the inhibition of cholinesterase activity. In contrast to the anticholinesterase effect, the bradycardic effect did not reach a plateau and continued to increase even at doses at which the cholinesterase inhibition was maximal. The maximal decrease in heart rate when the heart was still in sinus rhythm was by 81 +/- 13 bpm (49 +/- 7% of baseline), which was produced by a dose of 0.32 mg.kg-1. Edrophonium produced dose-dependent decreases in cholinesterase activity and heart rate, which were highly correlated (correlation coefficient r = 0.99, P < 0.0001). The ED50 of the reduction in heart rate (0.9 +/- 0.18 mg.kg-1) and cholinesterase activity (0.89 +/- 0.12 mg.kg-1) produced by edrophonium were similar. Moreover, the reduction in heart rate and cholinesterase activity produced by edrophonium reached a plateau at the same dose (6.4 mg.kg-1). At this dose, heart rate decreased by 22 +/- 2 bpm (14.6 +/- 0.9% of baseline). Compared to the bradycardia produced by stimulation of the vagus nerve, that produced by neostigmine was blocked by muscarinic antagonists at significantly lower doses while that produced by edrophonium was blocked at similar doses. CONCLUSIONS: The neostigmine-induced bradycardia is poorly correlated with cholinesterase inhibition compared to that produced by edrophonium, and has a higher sensitivity to muscarinic receptor antagonists compared to that produced by edrophonium or vagus nerve stimulation. These results are consistent with the hypothesis that the neostigmine-induced bradycardia is, in part, the result of neostigmine directly activating cholinergic receptors within the cardiac parasympathetic pathway. The bradycardia produced by edrophonium may be accounted for solely by an anticholinesterase action.

We studied the possible effects of repetitive (1-min interval) 50- and 100-Hz tetanic stimuli on 50-Hz and 100-Hz tetanic fade ratios (RF50HZ and RF100HZ). We also evaluated the sensitivity of the recorded responses to these two tests to assess residual neuromuscular block (isometric adductor pollicis mechanical activity), either during spontaneous recovery, or 15 min after neostigmine administration, in 22 adult anesthetized (thiopental, fentanyl, N2O/O2) patients receiving vecuronium. Two 50-Hz and two 100-Hz, 5-s duration, tetanic stimulations were randomly assessed at 1-min intervals: in a spontaneous (SPO) group (n = 11), when train-of-four (TOF) ratio spontaneously regained 0.7, and in a neostigmine (NEO) group (n = 11), 15 min after 40 micrograms/kg neostigmine was given intravenously at 25% return of control twitch tension. In the SPO group, when TOF ratio was 0.7, RF50HZ was 0.92 +/- 0.01 before and after subsequent tetanic stimulation, while RF100HZ was 0.48 +/- 0.05 and 0.47 +/- 0.05, respectively (not significant [NS]). In the NEO group, when TOF ratio was approximately 0.9, RF50HZ was 0.93 +/- 0.01 before and after subsequent tetanic stimulation, while RF100HZ was 0.80 +/- 0.02 and 0.78 +/- 0.02, respectively (NS). From patient to patient, both RF50HZ and RF100HZ were also identical. In conclusion, in patients receiving vecuronium, 1) 5-s, 50- and 100-Hz tetanic stimuli may be repeated without changes at 1-min intervals and, 2) in contrast to RF50HZ, recorded RF100HZ enables one to determine residual neuromuscular block during spontaneous recovery (P < 0.001) such as after neostigmine reversal (P < 0.05).

BACKGROUND: Since neostigmine was introduced for reversal of neuromuscular block, there has been controversy about the optimum dose for antagonizing neuromuscular block. The purpose of this study was to characterise recovery of neuromuscular transmission following a vecuronium-induced block 15 min after neostigmine administration using different stimulation patterns, and to determine the effects of different doses of neostigmine given at various pre-reversal twitch heights. METHODS: Adductor pollicis (AP) mechanical activity in response to low (0.1 and 2 Hz) and high (50 and 100 Hz) frequency stimulation, was recorded 15 min after 20, 40 and 80 micrograms/kg neostigmine, given to reverse a vecuronium-induced block at 10, 25 and 50% pre-reversal twitch height (TH). Fifty four ASA class I and II anaesthetised (methohexital, fentanyl, N2O/O2) young adult patients were studied and randomly allocated into 9 groups of 6 patients each. RESULTS: In contrast to twitch height (TH) and residual force after 50 Hz, 5 s tetanic stimulation (RF50Hz), the greater sensitivity of train-of-four (TOF) ratio and residual force after 100 Hz, 5 s tetanic stimulation (RF100Hz) points out the best reversal conditions (prereversal TH and the optimal neostigmine dose) (P < 0.001, two-way analysis of variance). The highest reversal scores (about 0.9 TOF ratio and RF100Hz) were obtained when 40 micrograms/kg of neostigmine was given at 25 and 50% TH. A 0.9 TOF ratio was also observed when 40 micrograms/kg of neostigmine was given at 10% TH, but, under these conditions, RF100Hz was only 0.6 (P < 0.05, Duncan test). CONCLUSION: To optimise the reversal action of neostigmine in order to obtain the highest neuromuscular transmission recovery (0.9 TOF ratio and RF100Hz) during a vecuronium-induced neuromuscular block, the 40 micrograms/kg dose has to be given at 25 to 50% recovery of TH.

To test if recovery of neuromuscular transmission is complete after the use of neostigmine under standardized conditions, we have measured adductor pollicis mechanical activity in response to 0.1 Hz (twitch height), train-of-four (TOF) and 100 Hz (RF 100 Hz) ulnar nerve stimulations. We studied 56 adult anaesthetized (thiopentone, fentanyl, nitrous oxide in oxygen) patients, allocated randomly to one of four groups (n = 14) to receive rocuronium (group Roc), vecuronium (group Vec), atracurium (group Atr) or pancuronium (group Pan). Recovery of neuromuscular transmission was studied for 15 min after neostigmine 40 micrograms kg-1 was given at 25% recovery of twitch height. Fifteen minutes after antagonism, the TOF ratio was 0.91 (SEM 0.01), 0.88 (0.02) and 0.92 (0.01) (ns), and RF 100 Hz was 0.78 (0.01), 0.79 (0.02) and 0.78 (0.01) (ns) respectively, in patients in groups Roc, Vec and Atr, respectively. In patients in group Pan, TOF ratio and RF 100 Hz were only 0.76 (0.01) and 0.33 (0.04) respectively (P < 0.01, one-way analysis of variance, Duncan's multiple classification range tests). In contrast with pancuronium, antagonism of rocuronium-, vecuronium- and atracurium-induced neuromuscular blocks produced a similar high degree of recovery of neuromuscular transmission.

BACKGROUND: Reversal of neuromuscular blockade induced with pancuronium, d-tubocurarine, or doxacurium is achieved using smaller doses of neostigmine in adults than in children. Also, pancuronium- and doxacurium-induced blockade is reversed with smaller doses of edrophonium in children than in adults. The purpose of this study was to compare the spontaneous and neostigmine- and edrophonium-assisted recovery of mivacurium-induced neuromuscular block in adults and children. METHODS: Fifty-four adults, aged 40.1 +/- 10.9 yr, and 54 children, aged 4.9 +/- 0.7 yr, physical status ASA 1-2, were studied during propofol/fentanyl/nitrous oxide anesthesia. A Datex relaxograph was used to monitor the electromyographic response of the adductor pollicis to train-of-four stimulation of the ulnar nerve every 10 s. After induction of anesthesia, 0.2 mg x kg(-1) intravenous mivacurium was administered followed by an infusion to maintain 90-95% T1 block. At the end of surgery, one of four doses of neostigmine (5, 10, 20, and 50 micrograms x kg(-1)) or edrophonium (100, 200, 400, and 1,000 micrograms x kg(-1)) or placebo was given, by random allocation, when T1 had recovered to 10%. Values of T1 and train-of-four were measured for 10 min. RESULTS: Spontaneous recovery proceeded more rapidly in children than in adults. At 10 min, T1 had recovered to 97 +/- 2% (SD) in children compared with 69 +/- 11% in adults and train-of-four to 84 +/- 5% versus 30 +/- 13% (P<0.0001). In children, 10 min after reversal, recovery of T1 and train-of-four was not different from control after edrophonium and was enhanced only by the larger doses of neostigmine. In adults, recovery was accelerated by both edrophonium and neostigmine. Five minutes after reversal, recovery was improved by either drug in adults and in children. CONCLUSIONS: Spontaneous recovery from mivacurium- induced neuromuscular block is more rapid in children than in adults. Ten minutes after attempted reversal, recovery is accelerated by edrophonium and usually by neostigmine in adults but not in children. Thus, when reversal is required, edrophonium may be preferred to neostigmine.

Twelve patients of elapid ophitoxaemia presented with neuromuscular paralytic features were given anticholinesterase (Neostigmine) in recommended dosage. In four of these patients, despite neuromuscular paralysis, no ASV was used. All these four patients survived. In eight patients, ASV was used; in three of whom it used in doses less than 50 units, yet patients survived. Of the remaining five, despite use of ASV in higher doses (more than 50 units), two succumbed to death. Eight patients required ventilatory support. Hence, in absence of any definite role of ASV in management of elapid ophitoxaemia (snake bite), use of anticholinesterase drugs alone, with good supportive care and prevention of likely complications, can result in satisfactory outcome.

The duration of clinical relaxation induced by vecuronium and reversal by neostigmine was studied in 40 patients with renal failure (RF) and 40 patients with normal renal function (NL) under general anesthesia. Patients were premedicated with flunitrazepam, and anesthesia commenced with fentanyl 1-2 micrograms/kg, thiopental 5-8 mg/kg, and vecuronium 0.1 mg/kg. Anesthesia was maintained with 60% nitrous oxide in oxygen, isoflurane 0.3%-1.0% end-tidal concentration, and 1 microgram/kg fentanyl every 20-30 min. Neuromuscular block was reversed by the administration of intravenous neostigmine 40 mg/kg at the time of reappearance of either two or four responses to the train-of-four (TOF) stimulation. Monitoring of neuromuscular function consisted of supramaximal TOF stimulation of the ulnar nerve and the evoked thumb response was registered using a force transducer. Spontaneous recovery time, reversal time, and the time to recovery of TOF ratio to 0.7 were recorded. RF did not prolong the vecuronium neuromuscular blocking effect, reversal was achieved at the same rate in NL as in RF, and the duration of reversal of neuromuscular blocking effect of vecuronium was not influenced by the time of administration of neostigmine. Therefore, the neuromuscular blocking effect of a tracheal intubating dose of vecuronium can be reversed at the same rate in patients with end-stage RF as in patients with normal kidney function.

Envenomation by the monocellate cobra (Naja kaouthia) is usually manifested clinically as neurotoxicity and local tissue necrosis. Treatment often requires administration of large quantities of antivenin, resulting in a high incidence of serum reactions. In cases where antivenin administration may be delayed for several hours or administration is contraindicated, the use of the anticholinesterase drug neostigmine may temporarily reverse the potentially lethal neurological effects of the venom. Detailed in this report is the case of immediate and dramatic reversal of envenomation symptoms following the administration of the anticholinesterase neostigmine methyl sulfate.

alpha 2-Adrenergic agonists are thought to produce analgesia, in part, by activating spinal acetylcholine release. The purpose of the current study was to examine the interaction between intrathecal neostigmine and epidural clonidine for analgesia and side effects in humans. METHODS: A total of 58 volunteers received an intrathecal injection of 5% dextrose in normal saline (D5NS) or neostigmine (50, 100, or 200 micrograms in D5NS), followed in 1 h by epidural saline or clonidine (computer-controlled infusion targeted to 50, 100, 200, or 400 ng/ml in cerebrospinal fluid) using an isobolographic design. Visual analog scale pain to a noxious cold stimulus, nausea, weakness, sedation, and other safety variables was measured before and at specified intervals after drug administration. RESULTS: The first 21 volunteers randomized to receive intrathecal hyperbaric neostigmine rather than D5NS received the drug while in the sitting position, and had none-to-minimal analgesia 1 h later. The remaining volunteers received the drug while in the lateral position, and demonstrated dose-dependent analgesia in the foot 1 h later. Epidural clonidine also caused dose-dependent analgesia. The combination of neostigmine and clonidine resulted in an additive enhancement for analgesia, but no enhancement of each drug's side effects, and a reduction in clonidine-induced hypotension. Neostigmine injected into subjects in the lateral position diminished clonidine-induced reductions in blood pressure and plasma norepinephrine. CONCLUSION: These results support enhancement of alpha 2-adrenergic analgesia by intrathecal neostigmine, but do not demonstrate synergy, as observed in animals. Lack of enhancement of side effects suggests this combination may be clinically useful.

Neostigmine antagonism after suxamethonium followed by mivacurium chloride bolus and infusion was studied. Thirty ASA group I or II patients were given mivacurium 0.15 mg/kg followed by infusion during nitrous oxide-enflurane-pethidine anaesthesia. Train of four (TOF) stimuli were applied to the ulnar nerve at the wrist and TOF twitch height and ratio measured by TOF-GUARD nerve stimulator. Mivacurium infusion was titrated to give a 90% block of first twitch height. Patients were randomized into two groups. Group I patients recovered from the mivacurium block spontaneously while Group II patients were given neostigmine 0.05 mg/kg and atropine 0.02 mg/kg. Time to reach train of four ratio (TOFR) of 25%, 50% and 70% were measured. This study demonstrated a mean infusion rate of 5.1 +/- 1.8 micrograms/kg/min to maintain a 90% neuromuscular block. In the spontaneous recovery group, time to reach TOFR of 25%, 50% and 70% were 9.3 +/- 2.7 min, 13.5 +/- 3.0 min and 16.7 +/- 3.0 min respectively while the corresponding times in the neostigmine group were 5.2 +/- 1.7 min, 10.9 +/- 2.2 min and 16.1 +/- 7.4 min respectively. There were significant differences in the time taken to TOFR of 25% (P < 0.0001) and 50% (P < 0.05) but no difference in the time taken for TOFR to return to 70%. We concluded that mivacurium is suitable for use in caesarean section despite a decrease in plasma cholinesterase activity. Neostigmine antagonism is not required as a routine.

PURPOSE: The aim of the study was to determine the optimum time for administration of neostigmine during recovery from atracurium-induced neuromuscular blockade. METHODS: The study comprised 103 patients anaesthetised with midazolam, fentanyl, thiopentone, halothane, and nitrous oxide. Relaxation was induced with atracurium 0.5 mg. kg-1 and maintained with supplements of 0.15 mg. kg-1. The ulnar nerve was stimulated with train-of-four (TOF) and double burst stimulation (DBS). Evoked MMG responses were recorded. Patients were randomized to spontaneous recovery (n = 20) or to assisted recovery by neostigmine (0.07 mg.kg-1) at varying intervals (6-50 min) from the last atracurium dose (n = 83). RESULTS: The reversal time (time from administration of neostigmine to TOF ratio 0.7) was always < 13 min, when T1 (first twitch in TOF) was detectable or when D1 (first twitch in DBS) was > 5%. Total assisted recovery time (time from last supplemental atracurium dose to TOF ratio 0.7) increased with increasing T1 and D1 twitch heights (P < 0.05). The curve fitted to the scattergram with total assisted recovery time vs time from last atracurium supplement to neostigmine administration decreased to reach a minimum after which it increased to approach the line of identity. The minimum of the curve (total assisted recovery time 30.7 min) was reached when neostigmine was given 18.6 min after last atracurium supplement. At this time the T1 and D1 twitch height averaged 4 and 8% respectively. If prolongation of the minimum total recovery time of 2.5% is accepted, neostigmine can be given at T1 and D1 twitch height values of 0 to 8% and 4 to 15%, respectively. CONCLUSION: The optimum time for neostigmine administration, taking both the reversal time and total recovery time into consideration, is when 0 < T1 < 8% or when 5 < D1 < 15%. Giving neostigmine at more profound degrees of blockade prolongs reversal time, while giving neostigmine later in the recovery phase prolongs total recovery time

Some anticholinesterase (anti-ChE) drugs induce airway smooth muscle contraction. Whether anti-ChE drugs stimulate muscarinic receptors in airway smooth muscle as well as nicotinic receptors in neuromuscular junction is unknown. Since there is a direct relationship between phosphatidylinositol (PI) response and airway smooth muscle contraction induced by muscarinic agonists, we examined the effects of neostigmine, physostigmine, pyridostigmine, and edrophonium on PI response in the airway smooth muscle. The rat tracheal slices were incubated in Krebs-Henseleit solution containing LiCl and [3H]myo-inositol in the presence of carbachol, anti-ChE, or none of them. [3H]inositol monophosphate (IP1), which is a degradation product of PI response, was counted with a liquid scintillation counter. Inositol monophosphate accumulation was stimulated by neostigmine, physostigmine, and pyridostigmine in a dose-dependent manner, but was not affected by edrophonium. These increases were completely inhibited by atropine. The results suggest that neostigmine, physostigmine, and pyridostigmine stimulate PI response in the airway smooth muscle, which would cause bronchoconstriction, while edrophonium does not affect PI response.

PURPOSE To examine the influence of anticholinesterase drugs neostigmine and edrophonium (which have different effects on plasma cholinesterase activity) administered for antagonism of neuromuscular block on the duration of action of mivacurium (a neuromuscular blocking drug metabolised by plasma cholinesterase). METHODS: This was a randomized study where mivacurium 0.15 mg.kg-1 was administered to a control group or after administration of neostigmine 40 micrograms.kg-1 or edrophonium 1 mg.kg-1 (n = 10 for each group) administered 10 min earlier for antagonism of atracurium-induced neuromuscular block. Neuromuscular block was measured by stimulation of the ulnar nerve in a train-of-four mode (TOF) and measuring the force of contraction of the adductor pollicis muscle. Baseline plasma cholinesterase activity was estimated before drug administration in all the groups and following anticholinesterase administration. RESULTS: The times to recovery of T1 (first response in the TOF) to 25 and 90% of control and of the TOF ratio to 0.7 after 0.15 mg.kg-1 of mivacurium were 47, 65 and 70 min in the neostigmine group; 25, 36 and 36 min in the edrophonium group and 17, 29 and 27 min respectively in the control group (P < 0.01). The plasma cholinesterase activity (PCHE) after neostigmine decreased from 6596 to 1959 U.L-1 (P < 0.001) but there was no change after edrophonium (6140 to 6396 U.L-1).
CONCLUSIONS: The duration of action of mivacurium is prolonged by previous administration of neostigmine and this is most likely to be due to inhibition of PCHE activity.

Antivenom in order to be effective in the treatment of coral snake accidents must be injected very soon after the bite owing to the rapid rate of absorption of the venom neurotoxins. As this is not always possible, other forms of treatment besides serotherapy must be employed to avoid asphyxia and death. Neostigmine and artificial respiration are used for this purpose. Neostigmine restores neuromuscular transmission if the venom-induced blockade results from a reversible interaction of its neurotoxins with the end-plate receptors. This is the mechanism of the neuromuscular blockade produced by the venom of M. frontalis snakes from centereastern and southern Brazil, and Argentine. Neostigmine is able, therefore, to antagonize the blockade, and has been shown to be very effective in the treatment of the experimental envenomation of dogs and monkeys. In the present communication, two cases of M. frontalis accidents treated with antivenom and neostigmine are reported. In both, neostigmine was successful in producing regression of the paralysis, confirming the effectiveness shown in the treatment of the poisoning induced in animals by M. frontalis venom.

BACKGROUND: The metabolic hydrolysis of mivacurium (and succinylcholine) is markedly impaired in the presence of hereditary or acquired defects of pseudocholinesterase. Clinical reports are conflicting as to the utility of anticholinesterases, in the reversal of mivacurium paralysis. In the current study, the role of exogenous cholinesterases and/or of anticholinesterase, neostigmine, in the reversal of deep mivacurium-induced paralysis, was studied. The rat phrenic-diaphragm preparation, in a fixed volume of Krebs solution, was chosen to eliminate the confounding effects on the dissipation of neuromuscular effects caused by hydrolysis, elimination, and redistribution of the drug. METHODS: In the phrenic-diaphragm preparation, mivacurium was administered to obtain >90% single twitch inhibition. Single twitch responses (0.1 Hz) were monitored for 60 min, after which the response to train-of-four stimulation was tested. The reversal of mivacurium by 0.5, 1.0, or 2.0 units/ml of (true) acetylcholinesterase, bovine pseudocholinesterase, or human plasma cholinesterase and by neostigmine, 0.1, 1.0, or 10.0 micrograms/ml tested. The efficacy of human plasma cholinesterase, 1 unit/ml in combination with each of the above neostigmine concentrations, also was examined. The reversal of succinylcholine-induced paralysis by the acetylcholinesterase, bovine pseudocholinesterase, or human plasma cholinesterase (1 unit/ml) alone and in the presence of neostigmine (10.0 micrograms/ml) was additionally tested as a positive control. A train-of-four ratio > 0.75 was considered adequate reversal. RESULTS: Acetylcholinesterase was a poor hydrolyzer of mivacurium, as bioassayed by reversal of paralysis. Bovine pseudocholinesterase in concentrations of 0.5 and 1.0 units/ml did not effectively reverse single twitch and train-of-four responses by 60 min, but bovine pseudocholinesterase (2 units/ml) and all concentrations of human plasma cholinesterase did. Neostigmine alone, tested at all concentrations, was an incomplete reversal drug. Clinical or therapeutic concentrations (0.1 and 1.0 micrograms/ml) of neostigmine did not, but pharmacologic concentrations (10 micrograms/ml) interfere with the efficacy of human plasma cholinesterase (1 unit/ml). Bovine pseudocholinesterase and human plasma cholinesterase equally reversed the effects of succinylcholine but acetylcholinesterase did not, whereas the addition of 10 micrograms/ml neostigmine to the enzymes inhibited the reversal of succinylcholine. CONCLUSIONS: Human plasma cholinesterase will reverse mivacurium more effectively than bovine pseudocholinesterase, but both will effectively reverse succinylcholine. Acetylcholinesterase has no effects on either relaxant. The anticholinesterase neostigmine was an incomplete reversal drug. Pharmacologic concentrations of anticholinesterases do, while clinical or therapeutic concentrations do not, completely inhibit the metabolic activity of pseudocholinesterases.

Drugs with a depolarizing action at the myoneural junction may cause a rise in intracranial pressure. Neostigmine, which is commonly used to reverse residual myoneural blockade, has a depolarizing action, and yet its effect on intracranial pressure is unknown. Lumbar cerebrospinal fluid pressure, which mirrors intracranial pressure, was determined in 12 patients undergoing cerebral aneurysm surgery. Cerebrospinal fluid pressure was measured during dense myoneural blockade and after its reversal with neostigmine. These effects on cerebrospinal fluid pressure were compared with those produced when the arterial partial pressure of carbon dioxide (PaCO2) rose from 4 to 5 kPa. After reversal of myoneural block, there was a small (non-significant) change in cerebrospinal fluid pressure from 3.6 to 4.3 kPa and a larger (significant) rise in cerebrospinal fluid pressure to 9.7 kPa when the PaCO2 was allowed to rise. In this group of patients, reversal of myoneural blockade with neostigmine causes no significant change in cerebrospinal fluid pressure.

Patients with generalized myasthenia gravis (MG) often have associated ventilatory muscle involvement. It is not known whether patients with isolated ocular muscle involvement have identifiable involvement of their ventilatory muscles. Most studies have assessed muscle involvement by measuring muscle strength; however, we hypothesized that measures of ventilatory muscle endurance may be more sensitive tests of ventilatory muscle involvement in myasthenia gravis. We studied 17 patients with myasthenia gravis (four with ocular involvement alone and 13 with varying degrees of generalized myasthenia gravis). Spirometry, ventilatory muscle strength (maximum inspiratory and expiratory pressures (MIP and MEP)) and endurance (2 min incremental threshold loading test) were measured before and 20 min after i.m. neostigmine. We compared the results with those of 10 normal controls. We found no difference between patients with isolated ocular involvement and controls. Ocular myasthenia gravis patients did not improve after neostigmine. The patients with generalized myasthenia gravis had reduced baseline ventilatory muscle strength (MIP 67 cmH2O (70% of predicted), MEP 86 cmH2O (50% of pred) and endurance (mean maximal load achieved = 246 g, mean pressure at highest load (P) = 19.4 cmH2O) compared with controls. After neostigmine, there was a significant increase in MIP in patients with generalized myasthenia gravis and a trend towards an increased MEP. As a group, the patients with generalized myasthenia gravis did not demonstrate a change in their ventilatory muscle endurance after neostigmine; however, there was considerable interpatient variability in response. We conclude that patients with isolated ocular MG have normal ventilatory muscle strength when tested conventionally.

We followed the recovery time course in 46 patients antagonized by neostigmine (0.036 mg kg-1) at different levels of vecuronium-induced neuromuscular blockade ranging from post-tetanic count 1 to train-of-four ratio 0.4 and in 15 patients during spontaneous recovery. Non-linear regression curve fit analyses showed that the optimal time for neostigmine administration was when the first twitch in the train of four (T1) was between 1% and 10%. Visual analyses of the scattergram in which the level of block, when neostigmine was given, was plotted against total recovery time (the time elapsed from administration of the last dose of vecuronium to train-of-four ratio 0.7) gave the same result. The reduction in total recovery time achieved by neostigmine was 32.6 min (SE diff. 6.0 min). To achieve the optimum effect, neostigmine must therefore be given 32.6 min plus the required time for peak effect of neostigmine (5.3-7.1 min), i.e. 37.9-39.7 min, before train-of-four ratio 0.7 is reached. During spontaneous recovery this corresponds to a T1 between 1% and 15%.

(1) To determine the time to peak effect of neostigmine (time to peak antagonism) during atracurium- or vecuronium-induced neuromuscular block; and (2) to determine the effect on time to peak effect of neostigmine during atracurium-induced neuromuscular block, when the dose of neostigmine is increased from 35 micrograms/kg to 70 micrograms/kg. DESIGN: Prospective, randomized clinical study. SETTING: Gynecologic operating room suite at a university hospital. PATIENTS: 45 ASA I and II women admitted for gynecologic laparotomy. INTERVENTIONS: Anesthesia was performed with thiopental sodium, fentanyl, halothane, nitrous oxide, and atracurium or vecuronium. Train-of-four (TOF) stimulation and mechanomyography were used to monitor neuromuscular transmission. Neostigmine was administered while a constant degree of neuromuscular block was maintained at a twitch height at a point between 4% and 11% of the control twitch height, using a continuous infusion of atracurium or vecuronium. The patients were randomized to three groups, with 15 patients in each group. Group 1 received atracurium block antagonized with neostigmine 35 micrograms/kg; group 2 received vecuronium block antagonized with neostigmine 35 micrograms/kg; and group 3 received atracurium block antagonized with neostigmine 70 micrograms/kg. MEASUREMENTS AND MAIN RESULTS: The degree of neuromuscular block at antagonism was similar in the three groups. Time to peak effect (mean +/- SD) on TOF ratio was significantly longer in Group 1 (9.7 +/- 3.0 minutes) versus Group 2 (6.6 +/- 1.4 minutes; (p < 0.05). The time to peak effect on TOF ratio during atracurium-induced block was reduced from 9.7 +/- 3.0 minutes to 6.3 +/- 2.0 minutes when the dose of neostigmine was increased from 35 micrograms/kg to 70 micrograms/kg (p < 0.05). The peak effect on TOF ratio was significantly greater in Group 3 compared with Group 1 (p < 0.05), while it was similar in groups 1 and 2. CONCLUSION: The time to peak effect of neostigmine 35 micrograms/kg is about 6 to 10 minutes when antagonizing a constant degree of atracurium- or vecuronium-induced neuromuscular block at a twitch height at a point between 4% and 11%. Even though the time to peak effect was longer with atracurium than with vecuronium, clinically significant differences between the antagonizing effect of atracurium versus vecuronium block were not demonstrated. The time to peak effect during atracurium-induced block decreased when the dose of neostigmine was increased from 35 micrograms/kg to 70 micrograms/kg.

Rapid sequence induction of anaesthesia necessitating the use of suxamethonium may occasionally be needed soon after antagonism of neuromuscular block with anticholinesterase agents. The onset and duration of action of 1 mg kg-1 of suxamethonium was recorded in groups of 10 patients each, 5 or 10 min after the administration of edrophonium 1 mg kg-1 or neostigmine 40 micrograms kg-1 given for the antagonism of atracurium-induced neuromuscular block. Plasma cholinesterase activity was measured before, and 5 and 10 min after the administration of the anticholinesterases. A further 10 patients received suxamethonium 1 mg kg-1 without prior atracurium or anticholinesterase administration to serve as controls. The onset of action of suxamethonium was significantly prolonged when administered 5 min after both anticholinesterases, compared to the control group (P < 0.01). Recovery of suxamethonium block was delayed significantly after neostigmine, compared to both the edrophonium and the control groups (P < 0.05-0.001). Plasma cholinesterase activity was significantly reduced with the use of neostigmine but not with edrophonium (P < 0.001).

Induction of c-Fos expression in the supraoptic nucleus (SON) of the rat hypothalamus by endogenous acetylcholine was examined by microinfusion of neostigmine, a cholinesterase inhibitor, into the nucleus to locally accumulate the spontaneously released acetylcholine from the cholinergic terminals in the SON. Double staining of the neurosecretory neurons with antiserum to Fos, the protein product of c-Fos, and antiserum to vasopressin or oxytocin was performed. Fos-like immunoreactivity was manifested in both the vasopressin neurons and oxytocin neurons following the microinfusion of neostigmine. Microinfusion of nicotinic agonist, nicotine, to the SON also induced Fos expression, but mainly in the vasopressin neurons. Microinfusion of muscarinic agonist, carbachol, induced Fos expression as well, but mostly in the oxytocin neurons.

The effect of bethanechol, neostigmine, metoclopramide, and propranolol on myoelectric activity of the ileum, cecum, and proximal loop of the ascending colon was determined in 6 healthy Jersey cows implanted with 8 pairs of bipolar electrodes. Assigned at random, each cow received each of 5 treatments in 3-day intervals. The treatments included bethanechol (0.07 mg/kg of body weight, SC), neostigmine (0.02 mg/kg, SC), metoclopramide (0.15 mg/kg, IM), DL-propranolol (0.2 mg/kg, IM), and 0.9% sodium chloride (NaCl) solution (20 ml, SC). All drugs were administered during early phase I of the migrating myoelectric complex in the ileum. Myoelectric activity was recorded for 4 hours after treatment, and data were analyzed for each hour separately. Bethanechol and neostigmine significantly (P < 0.05) increased the number of cecocolic spikes per minute per electrode, duration of cecocolic spike activity (%), and number of cecocolic propagated spike sequences per 10 minutes, relative to NaCl, during 1 or more hours of the recording period. The effect of bethanechol was more pronounced on duration of spike activity and number of propagated spike sequences, whereas neostigmine mainly increased the number of (uncoordinated) spikes. Metoclopramide and propranolol had no significant effect on cecocolic myoelectric activity, relative to NaCl. It was concluded that bethanechol and, less likely, neostigmine at the dosage used in this study may be suitable for medical treatment of cecal dilatation in cattle in which hypomotility of the cecum and proximal loop of the ascending colon has to be reversed. The potential advantage of bethanechol vs neostigmine for medical treatment of cecal dilatation is worth further evaluation.

The effectiveness of neostigmine 40 micrograms/kg for antagonism of two different levels of neuromuscular blockade, induced by a bolus dose of mivacurium 0.15 mg/kg, was studied in 45 patients. The patients were anaesthetized with thiopentone, fentanyl, nitrous oxide in oxygen, and enflurane. Neostigmine was administered at either 10% recovery of the twitch height (TH10) at the adductor pollicis muscle (n = 14) or upon reappearance of the first response at the orbicularis oculi muscle (OO1) after train-of-four (TOF) stimulation (n = 16), the latter representing a deeper degree of neuromuscular blockade. Fifteen of the 45 patients did not receive neostigmine (control group). Neostigmine administration at OO1 rather than at TH10 at the adductor pollicis muscle caused reversal of neuromuscular blockade to occur 8 min earlier and shortened the time to reach 25% recovery of the twitch height (TH25) at the adductor pollicis muscle by about 5 min, compared with the control group. However, the time needed to reach a T4/T1 ratio > or = 0.8 was similar in both the early and late neostigmine administration groups, being 9 min faster than in the control group. It can be concluded that there is no advantage in administering neostigmine at profound neuromuscular blockade to achieve clinically adequate recovery (T4/T1 ratio > or = 0.8). However, the time between injection of mivacurium and TH25 may be shortened by using neostigmine at profound neuromuscular blockade, a procedure which may be useful in case of unpredictably difficult intubation, since diaphragmatic movements usually reappear at TH25.

Pharmacokinetics of a very short-acting, a short-acting and two long-acting cholinesterase (ChE) inhibitors, edrophonium, neostigmine, pyridostigmine and ambenonium, respectively, were compared to elucidate the major determinant of their pharmacokinetics. No dose-dependency in pharmacokinetic behavior was observed within the range of 2-10 mumol/kg for edrophonium, 0.5-2 mumol/kg for pyridostigmine, 0.1-0.5 mumol/kg for neostigmine and 0.3-3 mumol/kg for ambenonium, respectively. Neostigmine has the shortest elimination half-life, and edrophonium, pyridostigmine and ambenonium follow in that. Four ChE inhibitors have similar Vdss values within the range of 0.3-0.7 l/kg, which is similar to the muscle/plasma concentration ratio of these drugs. The liver or kidney to plasma concentration ratio of all ChE inhibitors at 20min after i.v. administration ranged from 5 to 15. Small distribution volumes estimated from the plasma concentration profiles may reflect the distribution to muscle and to the extracellular space of other organs/tissues, while the rapid disappearance of ChE inhibitors from plasma may reflect the concentrative uptake to the liver and kidney.

Two patients were bitten by tunisian cobras (Naja haje haje). A few hours after the bite they developed generalized paralysis which progressed to respiratory paralysis. Electrophysiological features are similar to those of myasthenia gravis and exhibit decremental pattern after stimulation at a frequency of three per second. It is probable that cobra neurotoxins act postsynaptically. The neuromuscular blocking activity of snake-venom can be reversed by intravenous neostigmine.

Fluctuation analysis was used to estimate the mean single-channel conductance and the mean channel duration of opening. Miniature endplate currents (MEPCs) were measured with the voltage-clamp technique. The timing of endplate channel opening during the generation of the MEPC was estimated by a deconvolution method. Often all of the channels opened during the rise of the MEPC, but in about half of the examples some 10% of the channels opened after the peak. We studied the effects of acetylcholinesterase (AChE) inhibition with neostigmine, diisopropyl fluorophosphate (DFP) and fasciculin-2. With AChE largely inhibited, the number of channels opening increased as much as fourfold, largely by channels opening in the "tail" that follows the peak of the MEPC. The results were compared to models of MEPC generation. Models did not account well for the pattern of channel opening, particularly after AChE inhibition. In the presence of fasciculin-2, the addition of 2 microM (-)-vesamicol reduced the number of channels opening and shortened the period over which channels were open. One interpretation is that quantal ACh release is not almost instantaneous, but that some of the ACh is released over a period of a millisecond or more and that some of the release is blocked by (-)-vesamicol.

1. Neostigmine and BW284C51 induced concentration-dependent contractions in human isolated bronchial preparations whereas tetraisopropylpyrophosphoramide (iso-OMPA) was inactive on airway resting tone. 2. Neostigmine (0.1 microM) or iso-OMPA (100 microM) increased acetylcholine sensitivity in human isolated bronchial preparations but did not alter methacholine or carbachol concentration-effect curves. 3. In the presence of iso-OMPA (10 microM) the bronchial rings were more sensitive to neostigmine. The pD2 values were, control: 6.05 +/- 0.15 and treated: 6.91 +/- 0.14. 4. Neostigmine or iso-OMPA retarded the degradation of acetylcholine when this substrate was exogenously added to human isolated airways. A marked reduction of acetylcholine degradation was observed in the presence of both inhibitors. Exogenous butyrylcholine degradation was prevented by iso-OMPA (10 microM) but not by neostigmine (0.1 microM). 5. These results suggest the presence of butyrylcholinesterase activity in human bronchial muscle and this enzyme may co-regulate the degradation of acetylcholine in this tissue.

The protective action of 1,2,3,4-tetrahydro-9-aminoacridine (THA) against the long-lasting inactivation of acetylcholinesterase (AChE) and butyrylcholinesterase (BCHE) brought about by diisopropylfluorophosphate (DFP) and physostigmine, as well as by neostigmine in the case of AChE only, was evaluated by a dilution technique using Electrophorus electricus AChE and horse serum BCHE as target enzymes. In parallel experiments, the ability of physostigmine itself to protect these enzymes from DFP was evaluated and compared with that of THA. THA pretreatment was seen to prevent in a dose-dependent manner the inhibition of both AChE and BCHE. However, it was appreciably more potent towards AChE than towards BCHE. THA mean EC50 values for protecting AChE against 10, 40 and 100 microM DFP were 0.04, 0.16 and 0.45 microM, respectively; against 1 microM physostigmine the value was 1.8 microM and against 1.2 microM neostigmine it was 3.0 microM. The THA mean EC50 value for protecting BCHE against 3 microM physostigmine was 0.55 microM and the values for protecting against 3, 10 and 40 microM DFP were 1.5, 3 and greater than 10 microM, respectively. The protective action of THA was time independent: recovery of the maximal enzymic activity was immediate upon dilution. Unlike THA, the protective action of physostigmine developed progressively after dilution and was maximal within 3-4 (AChE) or 6-8 hr (BCHE). Under our experimental conditions, 0.3 microM physostigmine protected approximately 70% of AChE from 40 microM DFP and 5 microM physostigmine protected 9 and 47% of BCHE from 40 and 3 microM DFP, respectively. The results of this work suggest that THA exerts its protective action by shielding the active site of AChE and BCHE from the attack of the inactivating agents on account of its higher enzymic affinity, whereas the protective action of physostigmine against DFP takes advantage also of the carbamylation of the enzyme. These results are in line with the hypothesis that protection of AChE is the primary mechanism responsible for the antidotal action of THA against organophosphorus poisoning.

People with genetic variants of cholinesterase respond abnormally to succinylcholine, experiencing substantial prolongation of muscle paralysis with apnea rather than the usual 2-6 min. The structure of usual cholinesterase has been determined including the complete amino acid and nucleotide sequence. This has allowed identification of altered amino acids and nucleotides. The variant most frequently found in patients who respond abnormally to succinylcholine is atypical cholinesterase, which occurs in homozygous form in 1 out of 3500 Caucasians. Atypical cholinesterase has a single substitution at nucleotide 209 which changes aspartic acid 70 to glycine. This suggests that Asp 70 is part of the anionic site, and that the absence of this negatively charged amino acid explains the reduced affinity of atypical cholinesterase for positively charged substrates and inhibitors. The clinical consequence of reduced affinity for succinylcholine is that none of the succinylcholine is hydrolyzed in blood and a large overdose reaches the nerve-muscle junction where it causes prolonged muscle paralysis. Silent cholinesterase has a frame shift mutation at glycine 117 which prematurely terminates protein synthesis and yields no active enzyme. The K variant, named in honor of W. Kalow, has threonine in place of alanine 539. The K variant is associated with 33% lower activity. All variants arise from a single locus as there is only one gene for human cholinesterase (EC 3.1.1.8). Comparison of amino acid sequences of esterases and proteases shows that cholinesterase belongs to a new family of serine esterases which is different from the serine proteases.

The identity of a peptidase activity with human serum pseudocholinesterase (PsChE) purified to apparent homogeneity was demonstrated by co-elution of both peptidase and PsChE activities from procainamide-Sepharose and concanavalin-A--Sepharose affinity chromatographic columns; comigration on polyacrylamide gel electrophoresis; co-elution on Sephadex G-200 gel filtration and coprecipitation at different dilutions of an antibody raised against purified PsChE. The purified enzyme showed a single protein band on gel electrophoresis under non-denaturing conditions. SDS gel electrophoresis under reducing conditions, followed by silver staining, also gave a single protein band (Mr approximately equal to 90,000). Peptidase activity using different peptides showed the release of C-terminal amino acids. Blocking the carboxy terminal by an amide or ester group did not prevent the hydrolysis of peptides. There was no evidence for release of N-terminal amino acids. Potent anionic or esterase site inhibitors of PsChE, such as eserine sulphate, neostigmine, procainamide, ethopropazine, imipramine, diisopropylfluorophosphate, tetra-isopropylpyrophosphoramide and phenyl boronic acid, did not inhibit the peptidase activity. An anionic site inhibitor (neostigmine or eserine) in combination with an esterase site inhibitor (diisopropylfluorophosphate) also did not inhibit the peptidase. However, the choline esters (acetylcholine, butyrylcholine, propionylcholine, benzoylcholine and succinylcholine) markedly inhibited the peptidase activity in parallel to PsChE. Choline alone or in combination with acetate, butyrate, propionate, benzoate or succinate did not significantly inhibit the peptidase activity. It appeared that inhibitor compounds which bind to both the anionic and esteratic sites simultaneously (like the substrate analogues choline esters) could inhibit the peptidase activity possibly through conformational changes affecting a peptidase domain.

The protective action of eseroline--(3aS,8aR)-1,2,3,3a,8,8a-hexahydro-1,3 a, 8-trimethyl-pyrrolo[2,3-b]indol-5-ol--salicylate against (DFP) diisopropyl fluorophosphate and carbamate poisoning of cholinesterases (ChEs) has been examined in-vitro with human erythrocytes and purified preparations of electric eel acetylcholinesterase (AChE) and of horse serum butyrylcholinesterase (BCHE), and in-vivo using mice. Eseroline afforded 50% protection (ED 50) of erythrocyte AChE against inactivation by 1 microM DFP, physostigmine or neostigmine, at concentrations of 4.3, 22 and 23.5 microM, respectively, while for eel AChE protection against 10 and 30 microM DFP, 0.3 and 1 microM physostigmine and 1 microM neostigmine the eseroline ED 50 values were 0.3, 0.4, 0.7, 1.9 and 5.6 microM, respectively. On the other hand, up to 0.3 mM eseroline did not appreciably affect the inhibitory action of the same drugs on horse serum BCHE. Eseroline concentrations in the range 0.1-1 mM were able to reactivate 20-42% of erythrocyte AChE previously inhibited by 100 microM physostigmine, but failed to reactivate the DFP (10 microM)-pretreated enzyme to any extent. Finally, eseroline salicylate injected into mice (10 mg kg-1 s.c.) protected an average of 82 and 26% of the animals against lethal doses of DFP (7 mg kg-1 s.c.) and physostigmine sulphate (1 mg kg-1 i.p.) respectively, which were administered 15 min later. These results indicate that the protective activity of eseroline correlates well with its own anti-ChE profile, and that the effectiveness of the protection depends largely on the rate of AChE inhibition by the agents used to inactivate the enzyme.

Cholinesterase inhibitors are known to potentiate the effects of acetylcholine (ACh) and vagal stimulation on the myocardium. The studies presented here demonstrate that cholinesterase inhibitors (ChEI) also have activity in isolated atria in the absence of extrinsic cholinergic stimulation and that, depending on the ChEI, either indirect stimulation or direct blockade of cardiac muscarinic receptors can occur. Muscarinic agonists inhibit cyclic AMP formation in atria and the ChEIs physostigmine, neostigmine and echothiophate likewise produce a marked attenuation of isoproterenol-stimulated cyclic AMP accumulation The effect of physostigmine appears to result from muscarinic receptor activation by endogenous ACh as it is blocked by atropine. In contrast, the ChEI ambenonium does not stimulate but instead blocks muscarinic receptors coupled to cyclic AMP accumulation. Radioligand binding studies provide direct evidence that both ambenonium and demecarium are relatively potent muscarinic receptor antagonists, whereas physostigmine and other ChEI have little direct receptor activity. Physostigmine and ambenonium also have different effects on heart rate in vivo, the former potentiating and the latter apparently blocking vagal tone. The inhibition of cyclic AMP formation produced by physostigmine can be used as a measure of the concentration of endogenous ACh available at muscarinic receptor sites. Physostigmine blocks cyclic AMP formation in atria incubated in the absence of calcium or in the presence of tetrodotoxin, suggesting that endogenous ACh is spontaneously released in the absence of neuronal activity or depolarization-secretion coupling.

The inhibition of human motor endplate cholinesterase by anticholinesterase compounds was studied using isolated muscle membrane preparation. Ambenonium was most potent, and edrophonium was least potent in inhibiting motor endplate cholinesterase. The slope of the regression line for inhibition of motor endplate cholinesterase was greatest for ambenonium, and smallest for neostigmine and edrophonium. These compounds were less potent inhibitors of plasma cholinesterase. Ambenonium was more specific, and other compounds were less specific inhibitors of motor endplate cholinesterase. In myasthenic patients, these compounds produced adequate inhibition of motor endplate cholinesterase even in the presence of relatively mild plasma cholinesterase inhibition.

Human serum aryl acylamidase associated with serum cholinesterase was purified to homogeneity. Evidence for the identity of the two enzymes was based on co-elution profiles, co-purification in the different steps including affinity chromatography with constant ratios of specific activity and percentage recoveries, co-migration on gel electrophoresis, parallel inhibition by typical cholinesterase inhibitors and co-precipitation by antibody raised against the purified enzyme. Human liver aryl acylamidase was partially purified. Based on the elution profiles, purification data, inhibitory characteristics and gel electrophoresis it was concluded that aryl acrylamidase of liver was not associated with liver cholinesterase. More conclusive evidence for the non-association of the liver aryl acylamidase and cholinesterase came from their clear-cut separation on procainamide-Sepharose affinity chromatography. Both the serum and liver aryl acylamidase were compared with the purified erythrocyte aryl acylamidase associated with acetylcholinesterase. While the erythrocyte and serum aryl acylamidases showed some similarities in their sensitivities to amines like serotonin or tryptamine and choline derivatives, the liver enzyme was unaffected by any of these compounds. A notable observation was the activation by tyramine of the serum aryl acylamidase but not the erythrocyte and liver aryl acylamidases. The liver aryl acylamidase also differed from the other two in its relative insensitivity to inhibition by eserine, neostygmine and other cholinesterase inhibitors. Immunodiffusion and immunoprecipitation studies showed that the aryl acylamidases from the liver and erythrocytes were immunologically non-identical with the serum enzyme.

A steroid, the dibutyrate analogue of pancuronium bromide (9.8 X 10(-8)M), has been used as differential inhibitor in the study of the plasma cholinesterase variants. Pancuronium dibutyrate numbers have been measured for 190 individuals, and the mean values for six of the known genotypes, E1uE1u, E1uE1f, E1uE1a, E1fE1a, E1aE1a, and E1fE1f, have been calculated. Evidence is presented that a combination of the pancuronium dibutyrate number and the fluoride number give better resolution of the six genotypes than the combination of the pancuronium dibutyrate and the dibucaine number. This new differential inhibitor has real potential for revealing the probable existence of new genotypes.

The identity of the serotonin-sensitive aryl acylamidase with acetylcholinesterase from three diverse sources, namely sheep basal ganglia, human erythrocyte membrane and electric eel, was examined. Both the enzymes co-purified with constant ratios of specific activity from all the three sources by different affinity chromatographic techniques. The ratio of acetylcholinesterase to aryl acylamidase activity was highest for basal ganglia, less for erythrocyte and lowest for eel enzymes. Both the purified enzymes co-migrated on polyacrylamide gel electrophoresis either as a single species or multiple species under different conditions. Gel density gradient electrophoresis indicated identical migration rates of both the enzymes. Extraction of the enzymes from the three sources by different techniques of membrane disruption and subsequent gel filtration on Sepharose 6B showed multiple peaks of enzyme activity. Both the enzymes had identical elution profiles on Sepharose 6B gel filtration. All the enzyme peaks from Sepharose 6B on gel electrophoresis showed co-migration of the enzyme activities. Apart from inhibition by serotonin and acetylcholine the purified aryl acylamidases from all the three sources were potently inhibited by neostygmine, eserine and BW 284C51, all strong inhibitors of acetylcholinesterase. It is suggested that the serotonin-sensitive aryl acylamidase is identical with acetylcholinesterase.