Joan J. Kendig

Inhaled anesthetics and immobility: Mechanisms, mysteries, and minimum alveolar anesthetic concentration

Studies using molecular modeling, genetic engineering, neurophysiology/pharmacology, and whole animals have advanced our understanding of where and how inhaled anesthetics act to produce immobility (minimum alveolar anesthetic concentration; MAC) by actions on the spinal cord. Numerous ligand- and voltage-gated channels might plausibly mediate MAC, and specific animo acid sites in certain receptors present likely candidates for mediation. However, in vivo studies to date suggest that several channels or receptors may not be mediators (e.g., &ggr;-aminobutyric acid A, acetylcholine, potassium, 5-hydroxytryptamine-3, opioids, and &agr;2-adrenergic), whereas other receptors/channels (e.g., glycine, N-methyl-d-aspartate, and sodium) remain credible candidates.

Anesthetic actions within the spinal cord: contributions to the state of general anesthesia

The behavioral state known as general anesthesia is the result of actions of general anesthetic agents at multiple sites within the neuraxis. The most common end point used to measure the presence of anesthesia is absence of movement following the presentation of a noxious stimulus. The actions of general anesthetics within the spinal cord have been shown to contribute significantly to the suppression of pain-evoked movements, an important component of clinical anesthesia. Studies in the spinal cord are likely to increase our understanding of the pharmacology by which general anesthetics alter the transmission of somatomotor information. It now appears that the pharmacology responsible for the production of anesthesia is agent- and site-selective, and not the result of a unitary mechanism of action.

Hypothesis: Inhaled Anesthetics Produce Immobility and Amnesia by Different Mechanisms at Different Sites

Recent evidence supplies new insights regarding the two universal effects of inhaled anesthetics: 1) immobility in response to a noxious stimulus and 2) amnesia. We hypothesize that these two effects result from actions at separate molecular and anatomic sites and that they are produced by different mechanisms. We propose that inhaled anesthetics cause immobility in response to noxious stimuli by an action in the spinal cord at an interface between polar and nonpolar regions. Such a site might be an interfacial region adjacent to membranes or proteins. In contrast, we propose that production of amnesia occurs at a supraspinal site and occurs in a nonpolar environment. An example of such a nonpolar site could be the interior of a phospholipid bilayer or a hydrophobic pocket within a protein.

Propofol and Barbiturate Depression of Spinal Nociceptive Neurotransmission

Barbiturates are often described as non-analgesic or even hyperalgesic agents; the newer intravenous anesthetic agent propofol is said to be non-analgesic. Both propofol and barbiturates occupy sites on the GABAA receptor. The present study was designed to compare the effects of propofol and barbiturates on nociceptive-related neurotransmission in neonatal rat spinal cord; to search for actions that might be hyperalgesic; and to determine the extent to which propofol depression of nociceptive neurotransmission is mediated by GABAA receptors. The monosynaptic reflex, a slow ventral root potential (slow VRP) and the dorsal root potential (DRP) were recorded from isolated neonatal (1-5 days old) superfused rat spinal cords in response to electrical stimulation of a lumbar dorsal root. The slow VRP and the DRP are related to nociception. Propofol (0.5-10 microM), pentobarbital (1-10 microM), and thiopental (1-10 microM) reversibly depressed the slow VRP. Dose-response curves were monophasic and linear over this range. The monosynaptic reflex was unaffected. The GABAA agonist muscimol (0.2-1 microM) also depressed the slow VRP. Propofol and barbiturate slow VRP depression was antagonized by the GABAA antagonist bicuculline (1 microM). Propofol depressed the response evoked by direct application of substance P. The DRP is a GABAA-mediated depolarization of primary afferent nerve terminals that diminishes the effectiveness of nociceptive input. Propofol and thiopental increased electrically evoked DRP amplitude and increased the DRP evoked by application of muscimol. Both propofol and barbiturates thus depressed the nociceptive-related slow VRP and enhanced the antinociceptive DRP; their effective concentrations are at or close to the general anesthetic range for these agents. No anti-analgesic or hyperalgesic effect was observed. (ABSTRACT TRUNCATED AT 250 WORDS)

α2-Adrenoceptors inhibit a nociceptive response in neonatal rat spinal cord

Alpha 2-Adrenoceptors mediate analgesia in vivo. The present study explored the actions of the alpha 2-adrenoceptor agonists dexmedetomidine and clonidine on a nociceptive response in isolated neonatal rat spinal cord. Stimulation of a dorsal root generates a slow ventral root potential (slow VRP) at the corresponding ipsilateral ventral root. The slow VRP meets several criteria for a nociceptive response. Dexmedetomidine (10 nM) and clonidine (200 nM) depressed the slow VRP by approximately 80%. Dexmedetomidines action was approximately linear over the concentration range 0.5-500 nM, whereas clonidine (20 nM-5 microM) exerted biphasic effects. The profile of agonist and antagonist effectiveness characterized the receptor(s) as alpha 2-adrenoceptors; the subtype could not be identified as either alpha 2A or alpha 2B. Naloxone pretreatment partially blocked dexmedetomidines effect, suggesting a possible endogenous opiate involvement. Dexmedetomidine (0.5-2.0 nM) also depressed the VRP evoked by application of substance P to the cord, implicating postsynaptic as well as possible presynaptic actions. At high concentrations, dexmedetomidine (50-500 nM) depressed the monosynaptic reflex, probably through non-alpha 2-receptor(s). Results from the neonatal spinal cord correlate well with those from in vivo analgesia studies. They suggest an important direct spinal contribution to alpha 2-adrenoceptor-mediated analgesia.

Enflurane directly depresses glutamate ampa and NMDA currents in mouse spinal cord motor neurons independent of actions on GABAA or glycine receptors

BackgroundThe spinal cord is an important anatomic site at which volatile agents act to prevent movement in response to a noxious stimulus. This study was designed to test the hypothesis that enflurane acts directly on motor neurons to inhibit excitatory synaptic transmission at glutamate receptors. MethodsWhole-cell recordings were made in visually identified motor neurons in spinal cord slices from 1- to 4-day-old mice. Excitatory postsynaptic currents (EPSCs) or potentials (EPSPs) were evoked by electrical stimulation of the dorsal root entry area or dorsal horn. The EPSCs were isolated pharmacologically into glutamate N-methyl-d-aspartate (NMDA) receptor– and non-NMDA receptor–mediated components by using selective antagonists. Currents also were evoked by brief pulse pressure ejection of glutamate under various conditions of pharmacologic blockade. Enflurane was made up as a saturated stock solution and diluted in the superfusate; concentrations were measured using gas chromatography. ResultsExcitatory postsynaptic currents and EPSPs recorded from motor neurons by stimulation in the dorsal horn were mediated by glutamate receptors of both non-NMDA and NMDA subtypes. Enflurane at a general anesthetic concentration (one minimum alveolar anesthetic concentration) reversibly depressed EPSCs and EPSPs. Enflurane also depressed glutamate-evoked currents in the presence of tetrodotoxin (300 nm), showing that its actions are postsynaptic. Block of inhibitory &ggr;-aminobutyric acid A and glycine receptors by bicuculline (20 &mgr;m) or strychnine (2 &mgr;m) or both did not significantly reduce the effects of enflurane on glutamate-evoked currents. Enflurane also depressed glutamate-evoked currents if the inhibitory receptors were blocked and if either D,L-2-amino-5-phosphonopentanoic acid (50 &mgr;m) or 6-cyano-7-nitroquinoxaline-2,3-dione disodium (10 &mgr;m) was applied to block NMDA or &agr;-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid–kainate receptors respectively. ConclusionsEnflurane exerts direct depressant effects on both &agr;-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid and NMDA glutamate currents in motor neurons. Enhancement of &ggr;-aminobutyric acid A and glycine inhibition is not needed for this effect. Direct depression of glutamatergic excitatory transmission by a postsynaptic action on motor neurons thus may contribute to general anesthesia as defined by immobility in response to a noxious stimulus.

Isoflurane and an α2-Adrenoceptor Agonist Suppress Nociceptive Neurotransmission in Neonatal Rat Spinal Cord

Analgesia is an important component of general anesthesia. alpha 2-adrenoceptor agonists such as clonidine and dexmedetomidine are effective analgesics at the spinal level, and furthermore, they reduce the volatile anesthetic requirement. In order to probe a possible spinal-level contribution to general anesthetic-induced analgesia, the effects of dexmedetomidine were tested in an isolated spinal cord preparation. The effects of dexmedetomidine were compared with those of isoflurane, and dexmedetomidine-isoflurane interactions were explored. The test response was a nociceptive-related slow ventral root potential (slow VRP) recorded from the isolated neonatal rat spinal cord in response to electrical stimulation of a dorsal root. At 0.2-1.28 vol%, isoflurane reversibly depressed the slow VRP. At a lower concentration (0.14 vol%), isoflurane increased the slow VRP in three of five preparations. At 1.0-1.28 vol%, isoflurane also depressed the monosynaptic reflex. Recovery on washout usually was to a level greater than control. The N-methyl-D-aspartate (NMDA) receptor antagonist (DL)-2-amino 5-phosphonovalerate (10 microM) prevented the rebound to levels above control on isoflurane washout. The earlier components of the slow VRP were more sensitive to isoflurane than were the later. Dexmedetomidine (0.5-10 nM) depressed the slow VRP and had no effect on the monosynaptic reflex. The slow VRP depends on both substance P and glutamate NMDA-receptor-mediated neurotransmission; isoflurance and dexmedetomidine depressed responses to both substance P and NMDA. Although the two agents depress responses to the same neurotransmitters, there is no evidence that they act at the same cellular site(s). There was no significant interaction between dexmedetomidine and isoflurane. The results suggest that isoflurane exerts marked inhibitory effects on spinal neurotransmission, depressing both substance P and glutamate-mediated pathways. There is a possible biphasic effect on the NMDA receptor. To the extent that nociception depends on these neurotransmitters, isoflurane may be expected to exert profound analgesic effects at the spinal level. By blocking responses to strongly arousing stimuli, these effects may contribute to general anesthesia. Suppression of nociceptive neurotransmission at the spinal level may contribute to dexmedetomidines anesthetic-sparing properties as well as to analgesia by this agent.

Anesthetic effects on resting membrane potential are voltage-dependent and agent-specific

Membrane hyperpolarization (increase in resting potential) together with a conductance increase has been suggested as a common mechanism of anesthetic action. The current study compared the effects of halothane, enflurane, and isoflurane on resting membrane potential and conductance of hippocampal CA1 neurons in vitro. At 1 MAC, halothane produced significant (P less than 0.01) hyperpolarization (-2.8 +/- 1.3 mV, mean +/- SD) accompanied by a conductance increase (6.2 +/- 2.7%). Enflurane also produced a significant (P less than 0.001) hyperpolarization (-3.15 +/- 1.2 mV); however, this was accompanied by a conductance decrease (-4.5 +/- 1.5%). Isoflurane produced variable effects. Anesthetic-induced hyperpolarization was maximal in neurons with more negative initial resting potentials and was reduced by depolarization. Across agents, these relatively small changes in resting potential were not correlated with decreases in excitability as measured by synaptically evoked population spike depression. The results are not consistent with a common action of the three agents on a single ionic channel.

Relevant concentrations of inhaled anesthetics for in vitro studies of anesthetic mechanisms.

UNDERSTANDING how anesthetics act requires a synthesis of information from in vitro (molecular, receptor, and neuronal systems) and in vivo (whole animal) studies. Most investigators argue that only anesthetic concentrations required for clinical anesthesia (e.g., 0.2–2.0 minimum alveolar concentration [MAC]) are relevant to in vitro studies of anesthetic mechanisms. In a previous report in this journal, Eckenhoff and Johansson supplied several arguments for potential relevance to effects produced by any concentration, even concentrations far above the clinical range. In the current article, we conclude that only concentrations close to the clinical range are relevant to in vitro studies of anesthetic mechanisms. A Glossary of the several acronyms used in this article is supplied at the end of the article. Eckenhoff and Johansson offered a simple model in which the additive effects of anesthetics on different target molecules produce anesthesia. They found that if 10 receptors each have an EC50 of 1 unit (actual units are not relevant here) in vitro, then the combined in vivo effect occurs at a concentration of approximately 0.1 units, and the concentration–effect relation for the combined effect is steeper than for the individual in vitro concentration–effect relations. They found that this steepness approaches the steepness for concentration–effect relations for MAC. Such steepness is well illustrated with halothane: approximately 90% of patients move in response to incision at halothane concentrations below 0.72%, whereas only 10% move at concentrations exceeding 0.77% (fig. 1). The conclusions of Eckenhoff and Johansson may be questioned on two grounds. First, they assume that the total response of multiple effects is obtained by simple (parallel) addition. This has the perplexing result that the combined maximum effect is greater than 1.0. Second, their analyses fit a sigmoid curve to those data points less than 1.0 only. The resulting fit (forced to a maximum possible effect [Emax] 5 1.0) poorly describes the data. In fact, if the data are normalized to Emax 5 1, the slope equals that of a single receptor. The model in Appendix A considers sequential rather than parallel additivity and also predicts only small increases in steepness for multiple sites. Appendix B introduces the concept of threshold to better understand the relation between receptor and population sensitivities to anesthetics. The present article considers the concentrations relevant to in vitro studies of the mechanisms underlying one individual anesthetic effect, namely, immobilization. First, we examine the thesis that the additive effect of multiple receptors can produce concentration–effect responses similar to those found in the determination of MAC. We conclude that the steepness of the slope defining MAC results from the limited variations in individual responses to anesthetics and likely reflects a small number of target sites. Franks and Lieb reached a similar conclusion regarding the number of target sites based on results of studies of stereospecificity. Second, we consider whether in vitro studies of receptor effects should restrict the anesthetic concentrations applied to those that are clinically relevant. We conclude that they should. Receptor concentration–effect relations produce shallower curves than found for MAC determinations. For example, the concentration–effect relation for g-aminobutyric acid receptor type A (GABAA) and acetylcholine receptors goes from 10 to 90% of the maximum effect in one to two orders of magnitude (10to 100-fold) change in concentration (fig. 2). How might these in vitro shallow concentration–effect curves produce the in vivo steep concentration–effect curves for MAC? Eckenhoff and Johansson suggested that “the most plausible explanation for such highly conserved sensitivity to general anesthetics (i.e., the steepness of MAC concentration–effect curves) is that there are multiple contributing systems, each of which might be influenced to only a small degree by the anesthetic.” The steepness of the population curves that underlie MAC might be “explained by progressive, simultaneous actions at many targets of comparable sensitivity.” Eckenhoff and Johansson suggested that we can convert the relatively shallow receptor–concentration effect relations for a single receptor to steeper concentration–effect relations by adding the receptor–concentration effects of several different receptors. Fur* Professor, § Assistant Professor, Department of Anesthesia and Perioperative Care, University of California. † Professor, Department of Anesthesia and Perioperative Care, University of California. Current position: Vice President for Medical Affairs, Durect Corporation, Cupertino, California. ‡ Associate Professor, Department of Anesthesiology, State University of New York, Stony Brook, New York. \ Professor and Chairman, Department of Anesthesiology, Washington University, St. Louis, Missouri. # Professor and Head, ‡‡ Professorial Research Fellow, Biophysics Section, Blackett Laboratory, Imperial College of Science, Technology & Medicine, London, United Kingdom. ** Professor, §§ Research Fellow, University of Texas, Austin, Texas. †† Professor, Department of Anesthesia, Stanford University, Stanford, California.

Frequency-dependent Conduction Block: The Role of Nerve Impulse Pattern in Local Anesthetic Potency

The depth of local anesthetic-induced conduction block is modified by the frequency of impulse traffic in the nerve (frequency-dependent conduction block). The present study was designed to compare the frequency-dependent characteristics of a number of local anesthetics of different lipid solubilities. Two antiarrhythmic drugs, quinidine and propranolol, were also included. Frequency dependence was assessed by measuring the height of the compound action potential of the frog sciatic nerve in response to single stimuli and to stimuli presented repetitively at different frequencies. All the drugs tested showed marked enhancement of block at 40 Hz. Nerves treated with highly lipid-soluble agents (bupivacaine, tetracaine, etidocaine), two experimental compounds of low and very low lipid solubility (GEA 968 and QX-572, respectively), and the antiarrhythmic agents took longer to develop and to recover from the effects of stimulation than those treated with drugs of moderate lipid solubility (procaine, lidocaine, prilocaine, mepivacaine, and benzocaine). The effects of repetitive stimulation were apparent at lower frequencies for drugs in the former group than in the latter. The results support an important role for frequency dependence in the antiarrhythmic and local anesthetic properties of these drugs. They also reveal unexpected similarities between drugs at the high and low extremes of lipid solubility with respect to the time course of frequency-dependent blocking actions.