Informed consent for labor epidurals: a survey of Society for Obstetric Anesthesia and Perinatology anesthesiologists from the United States.

BACKGROUND: Ethicists agree that informed consent is a process rather than just simply the signing of a form. It should provide the patient with needed information and understanding to authorize a procedure. Essential elements of informed consent for women requesting labor epidurals include a description of the procedure, the risks and benefits, and alternative treatments for analgesia including the associated risks and benefits. The purpose of this pilot study was to determine practices and opinions of obstetric anesthesiologists regarding informed consent for parturients. METHODS: Questionnaires were sent to 885 anesthesiologists who were members of the Society of Obstetric Anesthesia and Perinatology based in United States institutions in 2002. RESULTS: Of the 885 questionnaires sent, 448 (51%) were returned with 47% from academic and 47% from private practice institutions. Forty-six percent worked as part of an obstetric anesthesia team; 51% worked in centers where there were >3000 deliveries/year. Sixty-eight percent suggested that "parturients in active labor are able to give informed consent for labor epidural analgesia." Thirteen percent recommend antenatal anesthesia consults for parturients inquiring about labor epidurals and 41% participated in childbirth classes. Responses did not differ significantly between physicians in academic vs. private practice. More obstetric team practices than non-team practices participated in childbirth education (54% vs. 30%, P < 0.0001). CONCLUSION: Despite the painful, stressful circumstances confronted by parturients, many respondents (76% in academic, 64% in private practice) thought that women in active labor are able to give informed consent.

Biophysical properties of Kv3.1 channels in SH-SY5Y human neuroblastoma cells.

Biophysical properties of delayed rectifier K channels in the human neuroblastoma SH-SY5Y were established using patch clamp recordings. The whole cell K+ conductance activated at membrane potentials positive to -20 mV. The midpoint of current activation was 9.6 +/- 5.1 mV, the equivalent charge was 3.7 +/-.6. Whole-cell currents inactivated slightly with time constants of 700 ms and 5 s. The K+ currents were sensitive to micromolar concentrations of TEA and 4-aminopyridine. RT-PCR experiments amplified a cDNA fragment specific for human Kv3.1 channels. Activation gating parameters in outside-out patches were shifted by approximately 14 mV in the hyperpolarizing direction.

The effects of general anaesthetics on ligand-gated ion channels.

The experimental effort that has been expended in investigating the effects of general anaesthetics on LGICs has been enormous over the past decade. Members of all three LGIC superfamilies have been examined using electrophysiological techniques. Anaesthetics that have been examined include volatile anaesthetics, gaseous anaesthetics, alcohols, i.v. anaesthetics and non-immobilizers. Obsolete anaesthetics (ether, cyclopropane, butane) have been used in order to increase the variability of the structure and polarity of experimental compounds. The tools of molecular biology have been used to make chimeric receptors and to make single-site mutations. Interestingly, this work has been taking place in parallel with efforts to understand the structure of these proteins. Anaesthetic research often stimulates structural research as well as vice versa. There are some common themes in the interactions between anaesthetics and the three superfamilies of LGICs. In many cases, anaesthetics have both inhibitory and potentiating effects on the channels. It is likely that the number of examples of this will increase when experiments are designed to look specifically for one or the other type of effect. So we must conclude that there are multiple binding sites for anaesthetics on LGICs. The degree of inhibition or potentiation is not easily predictable. In retrospect, this is not surprising when we consider that the sensitivity of a channel to anaesthetics can be altered by a single amino-acid mutation. The large structural differences between the cys-loop, glutamate-activated and P2X superfamilies do not lead to large differences in anaesthetic sensitivity. It is the smaller, almost insignificant, changes that do this. This observation that small changes may lead to large effects reinforces the idea that at least some of the interactions between anaesthetics and LGICs are direct drug-protein interactions that are not mediated by the lipids. This review has not addressed the question of whether the effects of anaesthetics seen on LGICs are relevant to anaesthesia. This question cannot really be answered at present. Although potent effects can be observed on the channels themselves, we have only begun to try to understand whether these effects are important for a synapse, a neuronal circuit or the function of an animal's nervous system. We have studied the trees; now we must go on to study the forest and the ecosystem.

The kinetics of inhibition of nicotinic acetylcholine receptors by (+)-tubocurarine and pancuronium.

Equilibrium conditions of neurotransmitter concentration and receptor binding are never achieved during synaptic transmission at the neuromuscular junction. Thus, it is important to determine the binding kinetics of drugs that act this synapse. Previous determinations of the dissociation rate of (+)-tubocurarine have produced inconsistent results ranging from 0.1 to 4000/s. Here, we used a direct approach to measure association (l(on)) and dissociation (l(on)) rates for two competitive antagonists (clinically used as nondepolarizing muscle relaxants), pancuronium and (+)-tubocurarine, at nicotinic acetylcholine receptors (nAChR). We made macroscopic current recordings from outside-out patches of BC3H-1 cells expressing embryonic mouse muscle nAChR. We used a three-tube rapid perfusion system to make timed applications of antagonists and acetylcholine to the patch. We made independent measurements of the equilibrium inhibition (IC(50)) and the kinetics of onset and recovery of antagonist inhibition at 20 to 23 degrees C. Rate constants were calculated from the predictions of a single (high-affinity) site model of competitive inhibition. For pancuronium: IC(50) = 5.5 +/- 0.5 nM (mean +/- S.D.), l(on) = 2.7 +/- 0.9 x 10(8) M(-1) s(-1), l(off) = 2.1 +/- 0.7/s [corrected] x 10(8)/s. For (+)-tubocurarine: IC(50) = 41 +/- 2 nM, l(on) = 1.2 +/- 0.2 x 10(8) M(-1) s(-1), l(off) = 5.9 +/- 1.3/s. The kinetic results are consistent with the equilibrium results in that l(off)/l(on) is in good agreement with the IC(50) values. All differences between the antagonists are significant at the p < 0.001 level. The higher affinity of pancuronium is caused by a faster association rate (2.2-fold) coupled with a slower dissociation rate (2.8-fold). The association rates of both antagonists are comparable with or greater than the association rate for acetylcholine binding to nAChR.

The kinetics of competitive antagonism by cisatracurium of embryonic and adult nicotinic acetylcholine receptors.

Competitive antagonists to nicotinic acetylcholine receptors are clinically used as muscle relaxants. Previously, we reported the kinetics of inhibition (in the absence of acetylcholine) by (+)-tubocurarine and pancuronium on embryonic receptors. Here, we examine cisatracurium, a commonly used muscle relaxant. Outside-out patches were equilibrated with cisatracurium before application of 300 microM acetylcholine. cisatracurium inhibited the initial peak current, but the decay of these currents displayed a pronounced biphasic behavior. The IC(50) value was 54 +/- 2 nM and 115 +/- 4 nM for adult and embryonic receptors, respectively. We designed a rapid perfusion system to apply or remove cisatracurium for various times before application of acetylcholine. We determined the association (embryonic, 3.4 +/- 0.4 x 10(8) M(-1) s(-1); adult, 1.8 +/- 0.3 x 10(8) M(-1) s(-1)) and dissociation (embryonic, 34 +/- 6/s; adult: 13 +/- 5/s) rates for cisatracurium. Association was 2.9- and 1.3-fold greater than that of (+)-tubocurarine and pancuronium, respectively. Dissociation was 6- and 16-fold higher than (+)-tubocurarine and pancuronium, respectively. These measurements correspond to dissociation of cisatracurium from receptors in the absence of acetylcholine. Physiologically, acetylcholine interacts with receptors equilibrated with antagonist. We developed a mathematical technique that removes the effect of desensitization and determined dissociation (embryonic, 52 +/- 9/s; adult, 33 +/- 5/s) in the presence of acetylcholine. These data suggest that presence of acetylcholine on one binding site of the receptor increases the dissociation rate of antagonist from the other binding site. We incorporated all of these rates into a computer simulation of a comprehensive 11-state Markov model. There was excellent agreement (without curve fitting) between simulated and experimental currents.

The Effects of isoflurane on acetylcholine receptor channels: 3. Effects of conservative polar-to-nonpolar mutations within the channel pore.

We performed macroscopic and single-channel current measurements on wild-type (WT) and two mutant muscle-type nicotinic acetylcholine (ACh) receptor channels transiently expressed in HEK-293 cells. The mutants contained polar-to-nonpolar substitutions at the 10' (alpha(2)S10'A beta T10'A gamma delta) and 6' positions (alpha(2)S6'A beta gamma delta S6'A) in the M2 pore region of the channel. We studied the behavior of these channels in the absence and presence of the volatile general anesthetic isoflurane. Both mutations changed the gating behavior of the channel. A comparison of the alpha(2)S10'A beta T10'A gamma delta mutant to WT receptors revealed faster desensitization kinetics, increased sensitivity to ACh, a higher efficacy for activation by the partial nicotinic agonist decamethonium, and a greater number of openings per burst. A comparison of the alpha(2)S6'A beta gamma delta S6'A mutant to WT receptors also revealed increased sensitivity to ACh and an increased burst duration at the single-channel level with ACh as agonist. The alpha(2)S10'A beta T10'A gamma delta mutation increased the sensitivity of the ACh receptor to isoflurane, whereas the alpha(2)S6'A beta gamma delta S6'A mutation did not. These changes were probably not caused by the differential effects of the mutation on channel gating and desensitization. The increased sensitivity of the alpha(2)S10'A beta T10'A gamma delta receptor to isoflurane is state-dependent; the mutation changes the affinity of the closed state but not that of the open state of the channel.

The noncompetitive inhibitor quinacrine modifies the desensitization kinetics of muscle acetylcholine receptors.

Quinacrine has been shown to act as a noncompetitive inhibitor of the nicotinic acetylcholine receptor (nAChR). However, its mechanism of action is still a matter of controversy. We analyzed in detail the action of quinacrine at both the single-channel and macroscopic current levels. The main effect of quinacrine is a profound concentration-dependent decrease in both the frequency of opening events and the duration of clusters elicited by high acetylcholine concentrations. Quinacrine also significantly increases (40-fold at 30 microM) the decay rate of macroscopic currents elicited by rapid perfusion of acetylcholine to outside-out patches. This decay is still well-described by a single exponential. Quinacrine has very little effect on the peak amplitude of the response, suggesting that it acts mainly on open channels. The recovery from desensitization after removal of acetylcholine is delayed in the presence of quinacrine. Results from both single-channel and macroscopic current recordings indicate that quinacrine increases the rate of nAChR desensitization and stabilizes the desensitized state. Interestingly, in equilibrium agonist-binding assays, quinacrine does not promote the typical high-affinity desensitized state. Thus, quinacrine seems to induce an intermediate state exhibiting the permeability but not the agonist binding properties of desensitization.

Inhibition of 5-HT3 receptors by propofol: equilibrium and kinetic measurements.

Patch-clamp/rapid solution exchange experiments as well as tracer ([14C]-guanidinium) influx measurements were applied to investigate effects of propofol on 5-HT3 receptor channels and compare the results with those obtained with pentobarbital. Currents induced by 30 microM 5-HT were recorded in outside-out patches from N1E-115 cells. Application of propofol 45 s before and during 5-HT application inhibited peak-currents and integrated current responses in a concentration-dependent manner (IC50 values=14.5 and 10.5 microM; Hill coefficients -1.5 and -1.3, respectively). The inhibitory effect of propofol in the current measurements was similar to the propofol-induced inhibition in tracer influx experiments in whole N1E-115 cells (Barann et al., 1993. Naunyn-Schmiedeberg's Archives of Pharmacology 347, 125-132). Pentobarbital-induced inhibition of 5-HT3 receptors in both patch-clamp (Barann et al., 1997. Neuropharmacology 36, 655-664) and tracer influx measurements indicated a lower potency and lower slope (IC50 values=130 and 55 microM; Hill coefficients -0.8 and -0.7, respectively) compared to propofol. Propofol, in contrast to pentobarbital, showed nearly the full potency when applied to the patches exclusively 45 s before 5-HT. Propofol was least effective when administered exclusively during 5-HT. The onset of inhibition of 5-HT-induced peak currents by propofol had a time constant of 220 ms, similar to the kinetics of 5-HT-induced desensitization.

Interactions of general anesthetics within the pore of an ion channel.

(1) We review the electrophysiological evidence that the ion channel pore is the site at which general anesthetics bind to inhibit muscle-type acetylcholine receptor channels. (2) The amphipathic character of a pore certainly offers a suitable environment for the binding of amphipathic anesthetics. (3) The absence of direct information about the binding sites of these rather non-specific drugs, forces us to rely on indirect information provided by kinetic experiments. (4) We also discuss the implications of these findings for the interaction of general anesthetics with other ion channels.

Mechanisms of barbiturate inhibition of acetylcholine receptor channels.

We used patch clamp techniques to study the inhibitory effects of pentobarbital and barbital on nicotinic acetylcholine receptor channels from BC3H-1 cells. Single channel recording from outside-out patches reveals that both drugs cause acetylcholine-activated channel events to occur in bursts. The mean duration of gaps within bursts in 2 ms for 0.1 mM pentobarbital and 0.05 ms for 1 mM barbital. In addition, 1 mM barbital reduces the apparent single channel current by 15%. Both barbiturates decrease the duration of openings within a burst but have only a small effect on the burst duration. Macroscopic currents were activated by rapid perfusion of 300 microM acetylcholine to outside-out patches. The concentration dependence of peak current inhibition was fit with a Hill function; for pentobarbital, Ki = 32 microM, n = 1.09; for barbital, Ki = 1900 microM, n = 1.24. Inhibition is voltage independent. The kinetics of inhibition by pentobarbital are at least 30 times faster than inhibition by barbital (3 ms vs. < 0.1 ms at the Ki). Pentobarbital binds > or = 10-fold more tightly to open channels than to closed channels; we could not determine whether the binding of barbital is state dependent. Experiments performed with both barbiturates reveal that they do not compete for a single binding site on the acetylcholine receptor channel protein, but the binding of one barbiturate destabilizes the binding of the other. These results support a kinetic model in which barbiturates bind to both open and closed states of the AChR and block the flow of ions through the channel. An additional, lower-affinity binding site for pentobarbital may explain the effects seen at > 100 microM pentobarbital.

Auscultation revisited: the waveform and spectral characteristics of breath sounds during general anesthesia.

Although auscultation is commonly used as a continuous monitoring tool during anesthesia, the breath sounds of anesthetized patients have never been systematically studied. In this investigation we used digital audio technology to record and analyze the breath sounds of 14 healthy adult patients receiving general anesthesia with positive pressure ventilation. Sounds recorded from inside the esophagus were compared to those recorded from the surface of the chest, and corresponding airflow was measured with a pneumotachograph. The sound samples associated with inspiratory and expiratory phases were analyzed in the time domain (RMS amplitude) and frequency domain (peak frequency, spectral edge, and power ratios). There was a positive linear correlation (R2 > 0.9) between inspiratory flow and sound amplitude in the precordial and esophageal samples of all patients. The RMS amplitude of the inspiratory and expiratory sounds was approximately 13 times greater when recorded from inside the esophagus than from the surface of the chest in all patients at all flows (p < 0.001). The peak frequency (Hz) was significantly higher in the esophageal recordings than the precordial samples (298 +/- 9 vs 181 +/- 10, P < 0.0001), as was the 97% spectral edge (Hz) (740 +/- 7 vs 348 +/- 16, P < 0.0001). In the adult population esophageal stethoscopes yield higher frequencies and greater amplitude than precordial stethoscopes. Quantification of lung sounds may provide for improved monitoring and diagnostic capability during anesthesia and surgery.

Interactions of general anaesthetics with single acetylcholine receptor channels.

We used single-channel recording techniques to study the effects of general anaesthetics on nicotinic acetylcholine receptor channels. Normally, these channels remain open for a few milliseconds. Anaesthetics induce three different patterns of channel activity. Ether causes the channel amplitude to be smaller and noisier than normal; isoflurane induces a flickery pattern in which openings occur in bursts of brief openings; propofol causes the channels to appear as isolated brief openings. These patterns can all be understood in terms of a model in which the anaesthetics bind directly to the channel protein and interrupt the flow of ions through the channel. The difference in pattern is determined by the duration of anaesthetic binding. Ether remains bound for the shortest period (< or = 0.01 ms), followed by isoflurane (0.5 ms) and propofol (> or = 2 ms). The anaesthetics may either be physically obstructing the pore of the channel or acting allosterically by inducing a new, non-conducting conformation of the channel.

Evidence for direct actions of general anesthetics on an ion channel protein. A new look at a unified mechanism of action.

BACKGROUND: Ion permeation through the nicotinic acetylcholine receptor channel is inhibited by general anesthetics. This inhibition could be mediated either by binding of anesthetic molecules to the channel protein itself or by the effects of anesthetics on the lipid environment of the protein. METHODS: Patch clamp recording techniques were used to investigate the effects of ether and propofol on acetylcholine receptor channels in outside-out patches from BC3H-1 cells. The kinetic and conductance properties of single channels were measured. A rapid perfusion system was used to make rapid changes in anesthetic concentration during patch clamp recording to determine the kinetics of inhibition by anesthetics. RESULTS: Ether, isoflurane (results from previous studies), and propofol produce distinct kinetic patterns of single acetylcholine receptor channel activity. Ether reduces the apparent current amplitude of channels, isoflurane induces flickering channel activity and propofol merely decreases the open time of the channel. The kinetics of inhibition are also different for these anesthetics. Ether (< 40 microseconds) is faster than isoflurane (300-600 microseconds) which is faster than propofol (> or = 2 ms). CONCLUSIONS: These diverse patterns can be interpreted in terms of a unitary mechanism in which the anesthetics interact directly with the channel protein. Each anesthetic is considered to bind to a site on the protein (perhaps, but not necessarily within the pore of the channel) and interrupt the flow of ions through the pore. Anesthetics have access to this inhibitory binding site even when the gate of the channel is closed. The pattern of channel activity induced by an anesthetic is determined by the frequency and duration of binding events.

Cooperative interactions between general anesthetics and QX-222 within the pore of the acetylcholine receptor ion channel.

To test the hypothesis that general anesthetics block nicotinic acetylcholine receptor channels by binding within the pore of the channel, we looked for competitive interactions between ether and QX-222 at the single channel current level. Experiments were performed on outside-out patches excised from BC3H-1 cells. QX-222 causes channels to flicker as it repeatedly binds within the pore of the channel and blocks the flow of current through the channel. Ether reduces the apparent unitary conductance of the channel. This effect of ether may be due to frequent, short-lived, unresolved, blockages of the channel. When both ether and QX-222 are applied, the effects of both drugs are seen on single channels. However, the duration of QX-222 blocking events are longer when ether is present; the duration of block is 0.89 +/- 0.06 ms with 30 microM QX-222 alone and 2.23 +/- 0.37 ms with 30 microM QX-222 + 20 mM ether (n = 5 +/- S.D.; -100 mV). Similar results are obtained when butanol is used in place of ether. We conclude that ether and QX-222 do not compete for a common binding site. Conversely, ether decreases the dissociation rate of QX-222. The simplest interpretation of these data is that the binding sites for ether and the aromatic moiety of QX-222 are distinct but close to each other; when ether is bound to its site, the binding of QX-222 is stabilized. We cannot, however, discount the possibility that ether stabilizes QX-222 by binding to a remote site and allosterically modifying the pore of the channel.

Effects of alcohols and volatile anesthetics on the activation of nicotinic acetylcholine receptor channels.

The n-alcohols butanol through nonanol and the volatile anesthetic ether increase the frequency of bursts of nicotinic acetylcholine (ACh) receptor channels induced by low concentrations of agonists. For example, 10 mM butanol increases the burst frequency induced by 0.2 microM ACh (a full agonist) and 1 microM decamethonium (a partial agonist) by 1.6-fold and 2.7-fold, respectively. An increase in burst frequency could arise from effects of the drug on agonist binding, channel gating, or desensitization. To distinguish among these alternatives, we measured the current response to rapid application of saturating concentrations of agonists. We found that 10 mM butanol increases the peak current induced by 100 microM decamethonium by 2-fold. In addition, 20 mM butanol and 3 mM pentanol both decrease the onset time of the current response to 10 mM ACh by about 40%. In contrast, ether does not increase the current response to 100 microM decamethonium and does not significantly change the onset time for 10 mM ACh. Neither ether nor butanol changes the degree of steady state desensitization induced by 0.2 microM ACh. We conclude that butanol and pentanol increase burst frequency by increasing the channel opening rate, whereas ether does so by increasing the agonist binding affinity of the ACh receptor.

The effects of isoflurane on acetylcholine receptor channels.: 2. Currents elicited by rapid perfusion of acetylcholine.

We studied the effects of the volatile anesthetic isoflurane on nicotinic acetylcholine (ACh) receptor channels using a technique for rapid perfusion of ACh to outside-out patches. Channels were activated by ACh, at concentrations ranging from 1 microM to 10 mM, and the macroscopic current flowing through tens or hundreds of channels was measured. Isoflurane reduced the peak current response to saturating concentrations of ACh, increased the current decay rate due to desensitization, and decreased the rate of recovery from desensitization. The effect of isoflurane on peak currents was concentration dependent; at 2% isoflurane, the peak current was reduced by half. The effect of isoflurane on the peak current induced by nonsaturating concentrations of ACh was smaller. We measured the onset and recovery of current inhibition by isoflurane, by rapidly applying and removing isoflurane to the patch within 100 microseconds. 2% isoflurane, currents were inhibited with a time constant of 200-300 microseconds and recovered with a time constant of 500-700 microseconds. We interpreted our results in terms of a kinetic model in which isoflurane binds directly to both open and closed channels (not necessarily within the pore of the channel) and stops the flow of ions through open channels. This model provides a quantitative explanation for the kinetic and equilibrium effects of isoflurane on peak currents activated by saturating concentrations of ACh. Our data support the idea that the flickering effect of isoflurane on single ACh receptor channels is caused by rapid binding and dissociation of isoflurane to an inhibitory binding site on the protein. The effects of isoflurane on the apparent affinity of ACh and on desensitization are not predicted by the model. These effects may arise from the binding of isoflurane to other sites, not necessarily on the protein itself.

Decamethonium is a partial agonist at the nicotinic acetylcholine receptor.

The efficacy of decamethonium as an agonist at the nicotinic acetylcholine receptor has never been determined. Here, we demonstrate how patch clamp recording during rapid perfusion of agonists to outside-out patches from BC3H-1 cells can be used to provide an unambiguous estimate of the efficacy of decamethonium. First, we obtain the decamethonium concentration-response relationship between 10 and 1,000 microM decamethonium. The maximum channel open probability is small (< 0.02) and occurs at about 100 microM. This suggests two alternative explanations: decamethonium is a poor agonist or decamethonium is an efficacious agonist but a potent channel blocker. To distinguish between these alternatives, we perfuse mixtures of decamethonium and acetylcholine to generate acetylcholine concentration-response curves in the presence of 30, 100, and 1,000 microM decamethonium. We use a model for activation and block of the acetylcholine receptor by both agonists to fit these data and determine the binding affinity, efficacy, and blocking affinity of decamethonium. We conclude that the efficacy of decamethonium is low, 0.016. Decamethonium is a true partial agonist.

Application of the one- and two-dimensional Ising models to studies of cooperativity between ion channels.

The Ising model of statistical physics provides a framework for studying systems of protomers in which nearest neighbors interact with each other. In this article, the Ising model is applied to the study of cooperative phenomena between ligand-gated ion channels. Expressions for the mean open channel probability, rho o, and the variance, sigma 2, are derived from the grand partition function. In the one-dimensional Ising model, interactions between neighboring open channels give rise to a sigmoidal rho o versus concentration curve and a nonquadratic relationship between sigma 2 and rho o. Positive cooperativity increases the slope at the midpoint of the rho o versus concentration curve, shifts the apparent binding affinity to lower concentrations, and increases the variance for a given rho o. Negative cooperativity has the opposite effects. Strong negative cooperativity results in a bimodal sigma 2 versus rho o curve. The slope of the rho o versus concentration curve increases linearly with the number of binding sites on a protomer, but the sigma 2 versus rho o relationship is independent of the number of ligand binding sites. Thus, the sigma 2 versus rho o curve provides unambiguous information about channel interactions. In the two-dimensional Ising model, rho o and sigma 2 are calculated numerically from a series expansion of the grand partition function appropriate for weak interactions. Virtually all of the features exhibited by the one-dimensional model are qualitatively present in the two-dimensional model. These models are also applicable to voltage-gated ion channels.