Neuroactive steroids, WIN-compounds and cholesterol share a common binding site on muscarinic acetylcholine receptors.

Endogenous neurosteroids and their synthetic analogues-neuroactive steroids-have been found to bind to muscarinic acetylcholine receptors and allosterically modulate acetylcholine binding and function. Using radioligand binding experiments we investigated their binding mode. We show that neuroactive steroids bind to two binding sites on muscarinic receptors. Their affinity for the high-affinity binding site is about 100 nM. Their affinity for the low-affinity binding site is about 10 µM. The high-affinity binding occurs at the same site as binding of steroid-based WIN-compounds that is different from the common allosteric binding site for alcuronium or gallamine that is located between the second and third extracellular loop of the receptor. This binding site is also different from the allosteric binding site for the structurally related aminosteroid-based myorelaxants pancuronium and rapacuronium. Membrane cholesterol competes with neurosteroids/neuroactive steroids binding to both high- and low-affinity binding site, indicating that both sites are oriented towards the cell membrane..

Biophysical study on the interaction of an anesthetic, vecuronium bromide with human serum albumin using spectroscopic and calorimetric methods.

The interactions between an anesthetic, vecuronium bromide (VB) and human serum albumin (HSA) have been investigated systematically by steady-state/time-resolved fluorescence, circular dichroism (CD), UV-vis absorption, Fourier transform infrared spectroscopy (FTIR), mass spectroscopy and differential scanning calorimetry (DSC) methods under physiological conditions. The fluorescence quenching observed is attributed to the formation of a complex between HSA and VB, and the reverse temperature effect of the fluorescence quenching has been found and discussed. Fluorescence analysis has proved that there is one classical binding site on HSA for VB with a relative weak binding constant of 1.07 × 10(4)M(-1) at 298 K. The primary binding pattern is determined by hydrogen bonding or van der Waals forces occurring in site I of HSA with ΔG°=-2.30 × 10(4)J mol(-1), ΔS°=-233 J mol(-1)K(-1) and ΔH°=-9.23 × 10(4)J mol(-1) at 298 K. VB could slightly change the secondary structure and induce unfolding of the polypeptides of protein. The DSC results provide quantitative information on the effect of VB on the stability of serum albumin. It is shown that VB can efficiently bind with HSA and be transported to the focuses needed.

Diabetes mellitus affects the duration of action of vecuronium in dogs.

OBJECTIVE: To compare the duration of action of vecuronium in diabetic dogs with a control group. STUDY DESIGN: Prospective clinical study. ANIMALS: Forty client-owned diabetic (n = 20) and non-diabetic dogs. METHODS: Dogs were considered free from other concurrent disease based on clinical examination and laboratory data. After pre-anaesthetic medication with acepromazine and methadone, anaesthesia was induced with intravenous (IV) propofol and maintained with isoflurane-nitrous oxide in oxygen. Neuromuscular blockade (NMB) was achieved with vecuronium, 0.1 mg kg(-1) IV and its effects recorded by palpation (pelvic limb digital extension) and electromyography (m. tibialis cranialis) of responses (twitches; T) to repeated train-of-four (TOF) nerve stimulation. Time to onset of NMB was the period between vecuronium injection and loss of fourth twitch (T4) in the TOF pattern recorded by EMG and palpation. Duration of NMB was defined as the time from drug administration to return of T1 by palpation (T1(tactile) ) and EMG (T1(EMG) ). Times to return of T2-4 were also recorded. Time from induction of anaesthesia to vecuronium injection was recorded. Heart rate, non-invasive mean arterial pressure, body temperature, end-tidal isoflurane and end-tidal CO(2) concentrations were recorded at onset of NMB and when T1(EMG) returned. Loss and return of palpable and EMG responses for diabetic and non-diabetic dogs were compared using t-tests and Mann Whitney U-tests. RESULTS: There were significant (p < 0.05) differences between diabetic and non-diabetic dogs for the return of all four palpable and EMG responses. Times (mean ± SD) for return of T1(tactile) were 13.2 ± 3.5 and 16.9 ± 4.2 minutes in diabetic and non-diabetic dogs respectively. There were no differences between diabetic and non-diabetic dogs in the time to onset of vecuronium with EMG or tactile monitoring. CONCLUSIONS AND CLINICAL RELEVANCE: The duration of action of vecuronium was shorter in diabetic dogs as indicated by both tactile and EMG monitoring.

The role of the amino acid residue at alpha1:189 in the binding of neuromuscular blocking agents to mouse and human muscle nicotinic acetylcholine receptors.

BACKGROUND AND PURPOSE: Nicotinic acetylcholine receptors (AChRs) are valuable therapeutic targets. To exploit them fully requires rapid assays for the evaluation of potentially therapeutic ligands and improved understanding of the interaction of such ligands with their receptor binding sites. EXPERIMENTAL APPROACH: A variety of neuromuscular blocking agents (NMBAs) were tested for their ability to inhibit the binding of [(125)I]alpha-bungarotoxin to TE671 cells expressing human muscle AChRs. Association and dissociation rate constants for vecuronium inhibition of functional agonist responses were then estimated by electrophysiological studies on mouse muscle AChRs expressed in Xenopus oocytes containing either wild type or mutant alpha1 subunits. KEY RESULTS: The TE671 inhibition binding assay allowed for the rapid detection of competitive nicotinic AChR ligands and the relative IC(50) results obtained for NMBAs agreed well with clinical data. Electrophysiological studies revealed that acetylcholine EC(50) values of muscle AChRs were not substantially altered by non-conservative mutagenesis of phenylalanine at alpha1:189 and proline at alpha1:194 to serine. However the alpha1:Phe189Ser mutation did result in a 3-4 fold increase in the rate of dissociation of vecuronium from mouse muscle AChRs. CONCLUSIONS AND IMPLICATIONS: The TE671 binding assay is a useful tool for the evaluation of potential therapeutic agents. The alpha1:Phe189Ser substitution, but not alpha1:Pro194Ser, significantly increases the rate of dissociation of vecuronium from mouse muscle AChRs. In contrast, these non-conservative mutations had little effect on EC(50) values. This suggests that the AChR agonist binding site has a robust functional architecture, possibly as a result of evolutionary 'reinforcement'.

[Prolonged paralysis after long-term administration of vecuronium in the intensive care unit].

A 51-year-old woman with renal failure requring hemodialysis received a total of 446 mg of vecuronium over a period of 4 days to help mechanical ventilation and developed generalized muscle weakness after its discontinuation. It took 2 wks to wean her from mechanical ventilation and 3 wks for her to be able to walk. She did not receive drugs which might alter neuromuscular transmission such as corticosteroid and aminoglycoside antibiotics. Electromyogram and nerve conduction studies were normal. We believe that the cause is the accumulation of vecuronium and active metabolites due to renal failure. The case suggests that we should minimize the duration of neuromuscular block in patients with renal failure by aggressive use of sedatives and analgesics and the optimization of ventilator settings.

A survey of combined epidural-propofol anesthesia with noninvasive positive pressure ventilation as a minimally invasive anesthetic protocol.

BACKGROUND: Combined epidural-propofol anesthesia with use of noninvasive positive pressure ventilation (NPPV) via the nose has been used routinely in our operating theaters. The purpose of this report was to present a survey of this anesthesia. MATERIAL/METHODS: 265 adult patients undergoing lower extremity or lower abdominal gynecological surgery during 1999 were examined. After epidural anesthesia, patients were given propofol infusion. NPPV was applied with an inspiratory/expiratory positive airway pressure of 14/8 cm H2O, a respiratory rate of 10 breaths/min, and oxygen delivery into the nasal mask resulting in a concentration of 40% or an inspiratory oxygen fraction of 0.35. Epidural anesthesia was continuously applied after surgery for postoperative pain relief. Various data related to the surgery or anesthesia were evaluated both on the day of surgery and on postoperative day 1. RESULTS: Of 265 patients, 3 patients could not receive our anesthetic protocol. Of the residual 262 patients, no patients showed serious clinical problems during anesthesia, excluding for hypotension, which was observed in 31-56% patients and was treated with ephedrine injection. Patients informed us of good analgesia (98%), feelings (78%) and dreams (47%). On postoperative day 1, postoperative analgesia and mood conditions were satisfactory. There were no patients complaining of intraoperative awareness. CONCLUSIONS: The principle of our anesthesia consists of epidural anesthesia, sole propofol infusion and noninvasive airway management, so as to provide an anesthetic technique with minimal invasiveness. Although airway maintenance by NPPV is not always suitable, our anesthesia is practicable for certain kinds of operations.

Unmasked residual neuromuscular block after administration of vecuronium for days.

IMPLICATIONS: Significant neuromuscular block may be present in patients who have received vecuronium for days.

A (17)O NMR study of peroxide binding to the active centre of bromoperoxidase from Ascophyllum nodosum.

The (17)O NMR of bromoperoxidase in Tris buffer at pH 8 treated with (17)O-enriched H2O2 reveals direct binding of peroxide to active site vanadium both in the symmetric and asymmetric modes, the latter possibly due to hydroperoxide. In addition, non-active site HVO2(O2)2(2-) is detected. The results are counter-checked with NMR data on peroxovanadium model compounds.

Should ICU patients receive muscle relaxants?

Neuromuscular blocking agents (NMBAs) are used in many critically ill patients, although their use is declining. NMBAs are designed for short term use in the operating theatre, and there are few studies in the critical care setting of either efficacy or safety, in particular their metabolism may be impaired by organ dysfunction. Weakness associated with critical illness is multifactorial, but in many cases is associated with myopathies and neuropathies. The possible role of NMBAs in the development of weakness is unclear, but there is no proven link between the use of NMBAs and neuropathy or myopathy.

Organic cation transport by rat liver lysosomes.

Hepatic organic cation transport has been characterized in rat liver plasma membrane vesicles, using the quaternary amine tetraethylammonium (TEA) as a model substrate. Sinusoidal TEA uptake is stimulated by an inside-negative membrane potential; TEA transport across the canalicular membrane is mediated by electroneutral organic cation-H+ exchange. Substrates for these transport processes include procainamide ethobromide (PAEB) and vecuronium, cationic drugs that undergo biliary excretion. Given the apparent absence of sinusoidal transport mechanisms able to generate high hepatocyte-to-blood organic cation concentration ratios, intracellular transport of organic cations may involve sequestration and concentration within acidified organelles. Therefore, the characteristics of TEA uptake were examined in isolated rat liver lysosomes that are acidified by a well-described H(+)-adenosinetriphosphatase (ATPase). Lysosomal uptake of [14C]TEA was a time- and ATP-dependent process, reaching steady state after 30-60 min. Steady-state [14C]TEA uptake was significantly reduced by omission of ATP and by addition of monensin, conditions that alter lysosomal pH and membrane potential gradients, and by the H(+)-ATPase inhibitors, N-ethylmaleimide and bafilomycin A. ATP-dependent lysosomal [14C]TEA uptake was significantly inhibited by PAEB, vecuronium, and other organic cationic substrates of canalicular TEA/H+ exchange. These findings demonstrate that rat liver lysosomes sequester certain organic cationic drugs, most likely via organic cation/H+ exchange driven by H(+)-ATPase. Canalicular organic cation/H+ exchange may reflect, in part, the exocytic insertion of this transporter from an intracellular compartment to this membrane domain.

Recovery of hepatic drug extraction after hypothermic preservation.

BACKGROUND: To determine whether liver preservation before transplantation impairs hepatic drug metabolism, hepatic extraction of drugs with different metabolic pathways (fentanyl, morphine, and vecuronium) in isolated rat livers was measured either immediately or after 24 h of hypothermia at 4 degrees C using a standard preservation-reperfusion sequence. METHODS: Isolated rat livers were perfused via the portal vein for 30 min to document initial viability. Test livers (n = 5) were perfused with iced Belzer solution, stored for 24 h at 4 degrees C, and flushed with 6% hetastarch. After hypothermic preservation for 24 h, or in control livers (n = 5) immediately after the 30-min perfusion, livers were perfused single-pass at a constant flow rate with solutions containing fentanyl, morphine, and vecuronium at 37 degrees C. Perfusate and bile samples were obtained at regular intervals for 64 min, after which liver tissue was harvested for analysis. Drug concentrations were measured using radioimmunoassay and gas chromatography. Metabolic capacity of the liver was estimated from the extraction fraction of each drug at steady-state. RESULTS: After warming to 37 degrees C, preserved livers consumed oxygen and produced bile at rates similar to that of control livers. Hypothermic preservation did not affect extraction of fentanyl and morphine. Vecuronium extraction was initially less in preserved livers, but this difference disappeared as the preserved livers returned to 37 degrees C (< 16 min). Biliary excretion and tissue concentrations of vecuronium were similar in each group. CONCLUSIONS: Hypothermic preservation does not significantly impair extraction of these drugs in this liver preservation model. If these results apply to human liver transplantation, little danger of drug accumulation exists during the early postoperative period if hepatic function is normal.

Spontaneous recovery or evoked reversal of neuromuscular block.

Recovery from the effects of muscle relaxants can occur either spontaneously by their metabolism in the body or by elimination via the normal excretion pathways, or by the administration of pharmacologic antagonists. The decision as to whether spontaneous recovery should be allowed to take place or pharmacologic reversal should be induced depends upon several factors, principal among them being the duration of action of the muscle relaxant used, its dose, and the time that is available. The recovery times of most relaxants, including atracurium and vecuronium, are such as to require antagonism if adequate recovery is to be attained quickly. An agent such as mivacurium may, however, allow complete spontaneous recovery to take place without the use of antagonists.

Characterization of transport in isolated human hepatocytes. A study with the bile acid taurocholic acid, the uncharged ouabain and the organic cations vecuronium and rocuronium.

The uptake and efflux of three categories of substrates were measured in isolated human hepatocytes and compared to those in rat hepatocytes. In addition, the extent to which the in vitro experiments quantitatively reflect liver function in vivo in both species was investigated. The anionic bile acid taurocholic acid was taken up by isolated human hepatocytes at a considerably lower rate than observed in isolated rat hepatocytes. Taurocholic acid uptake both in human hepatocytes and in liver plasma membrane vesicles showed sodium dependency. The uptake rate of taurocholic acid in isolated hepatocytes of both species was quantitatively compatible with the reported liver clearance of the bile acid in vivo. Ouabain uptake rate in isolated human hepatocytes was lower than in rat hepatocytes. This species difference was in accordance with pharmacokinetic studies in vivo on hepatic clearance of ouabain in man and rat. Uptake of vecuronium into human hepatocytes was about a factor of 10 lower than that in rat hepatocytes. Uptake into and efflux from human hepatocytes was comparable for the two short acting muscle relaxants vecuronium and rocuronium. Since distribution to the liver is considered to be a major factor in termination of action of vecuronium and rocuronium these observations were in line with the human pharmacokinetic profiles. In conclusion, the uptake rate of the studied model compounds in human hepatocytes appeared to be lower than that in rat hepatocytes. These observed transport rates reflected the relative hepatic transport rates observed in these species in the intact organism, but the absolute values in both species for some substrates may have been somewhat lower than calculated from in vivo data. It is concluded that transport studies in isolated hepatocytes are suitable for comparative drug transport studies, but are less precise in the prediction of quantitative membrane transport.

Antagonism of vecuronium by one of its metabolites in vitro.

The neuromuscular-blocking agent vecuronium bromide undergoes hydrolysis to three pharmacologically active metabolites (3-desacetyl, 17-desacetyl and 3,17-desacetyl vecuronium) which might modify the neuromuscular-blocking action of their parent compound. In order to elucidate the possible role of the interaction between vecuronium and its metabolites in the complications reported after long-term use of vecuronium in intensive care unit (ICU) patients, the relative potency of vecuronium, 3-desacetyl and 3,17-desacetyl vecuronium was determined in the rat hemidiaphragm in vitro and the mode of interaction of the above-mentioned compounds investigated. Dose-response relationships were established for each substance alone and for combinations of vecuronium with its metabolites. The relative potency at the EDmax50 levels (% maximal effect) were in the order of 1:1.2:27 for vecuronium, the 3-desacetyl derivative and the 3,17-desacetyl derivative, respectively. The mode of interaction characterized by isobolographic and algebraic (functional) analysis showed vecuronium and 3-desacetyl vecuronium to interact in an additive fashion while the combined effect of the parent compound and its 3,17-desacetyl derivative was less than additive, indicating antagonism.

Sequestration of organic cations by acidified hepatic endocytic vesicles and implications for biliary excretion.

A number of cationic amine drugs that are taken up by liver and excreted into bile may accumulate in acidified intracellular organelles such as lysosomes and endosomes. These studies were undertaken to assess directly the uptake and accumulation of three types of model organic cationic amines by endocytic vesicles, and the role of vesicle acidification in this process. Uptake of tubocurarine (TC), vecuronium and tributylmethylammonium (TBuMA) by purified rat liver multivesicular bodies (MVB) (prelysosomal endocytic vesicles) was dependent upon MgATP, time and drug concentration. After 60 min, 52 to 81% of MVB cation content was dependent upon vesicle acidification (due to an electrogenic proton pump), but not upon an interior positive vesicle membrane potential. Nineteen to 42% of MVB cation content appeared due to binding to MVB membranes or to internal lipoproteins. Vesicle-to-medium ATP-dependent apparent concentration ratios for these three cations were 3.3 to 51. MVB uptake of these cations resembled uptake of methylamine, a tertiary amine known to distribute across organellar membranes according to pH gradients. By contrast, MVB uptake of the lipophilic quaternary amine methyldeptropine was not dependent upon MgATP or on development of MVB pH or membrane potential gradients. In further studies, TC, vecuronium and TBuMA were rapidly taken up by the isolated perfused rat liver and excreted in bile. Exposure to 250 mciroM primaquin (which partially alkalinized acidic endosomes and lysosomes) reduced accumulation of [3H]vecuronium in a lysosomal fraction by 23%, decreased perfusate disappearance of TC and TBuMA, but not of vecuronium, and decreased biliary appearance of all three cations. These studies suggest that acidified intracellular organelles sequester certain organic cationic drugs, possibly via a drug/proton antiporter, and/or diffusion followed by intravesicular protonation and trapping of tertiary amines. However, attempts at partial displacement of these drugs, accomplished through partial vesicle alkalization by primaquin, decreased excretion of TC, vecuronium and TBuMA, perhaps reflecting the small functional size of the displaceable organellar drug compartment and/or competition between primaquin and the organic cations for membrane transport processes.

Drug transport, viability and morphology of isolated rat hepatocytes preserved for 24 hours in University of Wisconsin solution.

Isolated hepatocytes are a valuable tool to study liver functions. Suitable methods to preserve the isolated cells with good maintenance of viability and functions are crucial to extend experiments with hepatocytes from a single isolation over 2 or more consecutive days. We investigated whether University of Wisconsin (UW) solution, which was designed to preserve organs for transplantation, is also suitable for preservation of isolated rat hepatocytes. Viability, as determined by Trypan blue exclusion and reduction of the tetrazolium 3(4,5-dimethyl-thiazoyl-2-yl)2,5 diphenyltetrazolium bromide to a purple formazan, morphological appearance using electron microscopy, ATP levels and uptake and storage of three model drugs ([3H]vecuronium, [3H]taurocholic acid and [3H]ouabain) were determined directly after isolation and after 22 hr of storage in UW solution at 0-4 degrees. The present study shows that cold storage of rat hepatocytes for 22 hr in UW can be performed without significant loss of viability and with maintenance of proper morphology, cellular ATP and transport functions. In contrast, after storage in Krebs-Henseleit buffer the normal morphology, ATP content and transport functions were strongly affected. These results imply that hepatocytes from a single isolation and stored in UW solution can be used for experiments on 2 consecutive days.

Binding studies on two functional cardioselective antimuscarinic compounds.

Vecuronium and himbacine are antimuscarinic compounds which in functional studies exhibited a ca 6- and 10-fold higher potency at cardiac muscarinic receptors than at ileal muscarinic receptors. However in binding studies both compounds failed to differentiate between [3H](-)-QNB binding sites in guinea-pig atrial and ileal muscle homogenates. In the latter experiments, the dissociation constants of vecuronium in atria and ileum and that of himbacine in ileum were lower than the values determined functionally. The basis for the lack of cardioselectivity in binding studies is not known. These compounds add to the list of functional cardioselective muscarinic receptor antagonists that failed to display selectivity in binding studies with [3H](-)-QNB.