Volatile anesthetics and glutamate activation of N-methyl-D-aspartate receptors.

Several studies have indicated important functional interactions between volatile anesthetics and the N-methyl-D-aspartate (NMDA) class of glutamate receptors. In the present study, we examined the effects of diethyl ether, chloroform, methoxyflurane, halothane, enflurane, and isoflurane on (1) glutamate activation of the NMDA receptor complex, including glycine reversal of anesthetic action, as revealed by [3H]-(5R, 10S)-(+)methyl-10,11-dihydro-5H-dibenzo [a,d]cyclohepten-5,10-imine, dizocilpine (MK-801) binding to the cation channel, and (2) [3H]cis-4-( phosphonomethyl)piperidine-2-carboxylic acid (CGS 19755) binding to the glutamate recognition site of the NMDA receptor In agreement with previous studies, glutamate increased the binding of 1 nM [3H]MK-801, measured after a 1-hr incubation at 37 degrees, by up to several hundred fold. This stimulation was blocked by glutamate antagonists and potentiated by glycine with an EC50 of approximately 0.03 muM. Glycine also had a direct stimulatory effect on [3H]MK-801 binding at much higher concentrations ( > or = 10 muM). All of the anesthetics examined depressed glutamate stimulation of [3H]MK-801 binding in a concentration-dependent manner with the following order of potency: halothane > or = enflurane > methoxyflurane > chloroform > diethyl ether. This inhibition of [3H]MK-801 binding was observed at concentrations that are routinely attained in the cerebrospinal fluid during surgical anesthesia. Moreover, the inhibition was reversed rapidly following removal of the anesthetics from the assay medium. Inclusion of glycine in the incubation medium markedly attenuated anesthetic-induced inhibition of glutamate-sensitive [3H]MK-801 binding with an EC50 of between 0.1 and 1 muM. Thus, this reversal by glycine correlated with its potentiating as opposed to its direct stimulatory, effect on NMDA receptors. Anesthetic inhibition of [3H]MK-801 binding could not be overcome by raising the glutamate concentration (i.e. the interaction did not appear to be competitive with respect to glutamate) unless glycine was present. Binding of [3H]CGS 19755 to the glutamate recognition site was also inhibited by each of the anesthetics examined. However, with the exception of chloroform, all of the anesthetics were more potent inhibitors of glutamate-stimulated [3H]MK-801 binding than they were of [3H]CGS 19755 binding. [3H]CGS 19755 binding saturation curves in the presence of halothane and enflurane indicated a decrease in the density of [3H]-CGS 19755 binding sites with no change in binding affinity (i.e. the inhibition did not appear to be competitive). These findings support the idea that anesthetic drugs disrupt NMDA receptor transmission through multiple allosteric effects on the receptor-channel activation mechanisms and the glutamate binding site.

Volatile anesthetics inhibit NMDA-stimulated 45Ca uptake by rat brain microvesicles.

We have previously shown that volatile anesthetics inhibit glutamate-stimulated [3H]MK-801 binding to the ionophore of NMDA receptor complexes in rat brain. In the present study, we examined the influence of enflurane and halothane on NMDA-stimulated 45Ca uptake by a microvesicle fraction isolated from rat brain. NMDA stimulated 45Ca uptake (30 sec) by rat brain microvesicles by up to 70% with an EC50 of 1.4 +/- 0.5 microM. The NMDA-stimulated 45Ca uptake was inhibited by MK-801 and D-AP-5 with IC50's of approximately 10 microM. Enflurane and halothane inhibited 45Ca uptake stimulated by 100 microM NMDA by as much as 60-80% with IC50's of 0.2-0.3 mM, concentrations achieved during routine clinical use. Basal 45Ca uptake measured in the absence of agonist was not affected by the anesthetics. Glycine did not affect the level of NMDA-stimulated 45Ca uptake, but markedly reduced the inhibition of uptake caused by enflurane and halothane. Preincubation of microvesicles with NMDA resulted in a desensitization of NMDA-stimulated 45Ca uptake, with a t1/2 of approximately 20 sec. Enflurane and halothane diminished both the extent and rate of development of this desensitization, as did glycine. These findings support the idea that volatile anesthetic interference with neurotransmission at NMDA receptor complexes contributes to the development of the anesthetic state.

Influence of halothane on the interactions of serotonin1A and adenosine A1 receptors with G proteins in rat brain membranes.

The influence of halothane on the interactions of 5-HT1A and adenosine A1 receptors with G proteins was determined by monitoring the guanine nucleotide sensitivity of agonist binding to these receptors. Halothane inhibited the binding of radiolabeled agonists to 5-HT1A and adenosine A1 receptors by up to 30%, but only at concentrations considerably greater than those necessary for the maintenance of the anesthetic state. The sensitivity of high-affinity agonist binding to a guanine nucleotide (guanylyl-5'-imidodiphosphate) was not affected by halothane, indicating no disruption of receptor-G protein coupling. Thus, it appears that the ability of halothane to disrupt receptor-mediated signal transduction by interference with receptor-G protein interactions is receptor specific.

Disruption of muscarinic receptor-G protein coupling is a general property of liquid volatile anesthetics.

The influences of 3 volatile anesthetics, chloroform, enflurane and isoflurane, on muscarinic acetylcholine receptors in rat brainstem were determined. Each of the volatile anesthetics increased [3H]methylscopolamine [( 3H]MS) binding affinity, but did not affect the number of [3H]MS binding sites. Carbamylcholine affinity for brainstem muscarinic receptors was not altered after equilibration of brainstem membranes with any of these anesthetics. The ability of guanine nucleotides to depress the high affinity binding of two agonists, carbamylcholine and [3H]oxotremorine-M, was decreased or eliminated after equilibration of brainstem membranes with any of the anesthetics. In each of these actions, these anesthetics resemble halothane and diethyl ether. These results indicate that interference with muscarinic receptor-G protein interactions is a common property of liquid volatile anesthetics and may represent a general mechanism for the disruption of signal transmission between cells during anesthesia.

Anesthetic effects on muscarinic signal transduction.

A wealth of pharmacological and physiological evidence has established that anesthetics disrupt synaptic transmission at muscarinic and other synapses. The sequence of molecular events precipitated by agonist binding to the receptors is under intense scrutiny. It appears that at the majority of synapses G proteins serve to mediate the transfer of information from receptors to intracellular mechanisms. The major exception to this scheme is the situation in which an ion channel is incorporated directly in the receptor structure. Binding of an agonist to these receptors produces a conformational change in the receptors which opens an intrinsic ion channel. This situation occurs in nicotinic acetylcholine gamma-amino butyric acid type A (GABAA, and 5-hydroxytryptamine type 3 (5-HT3) receptors). Assays have been developed to evaluate several steps in the cascade of events involved in synaptic signal transduction, and these assays have been employed to determine the step at which anesthetics act to disrupt synaptic transmission. We have demonstrated that several volatile anesthetics alter the interaction of muscarinic receptors with transducer G proteins. Ligand-binding experiments suggest that receptor-G protein complexes are stabilized, thereby disrupting G protein GTPase activity and muscarinic control of cellular activity. This "stabilization" does not appear to involve an inhibition of guanine nucleotide binding, the proximal event in receptor-G protein dissociation. Two possibilities warrant further consideration: (1) that GDP release from inactive G protein trimers, which is normally catalyzed by the receptor, is inhibited, and (2) that receptor-G protein complexes fail to dissociate even in response to GTP binding. We are currently examining these possibilities using purified G proteins and receptors in reconstituted systems.

Diethyl ether effects on muscarinic acetylcholine receptor complexes in rat brainstem.

The influence of diethyl ether on muscarinic acetylcholine receptor-G protein interactions was studied using membranes isolated from rat brainstem. Membranes were equilibrated with diethyl ether (0.5 to 10%) for 20 min before, and then during, the binding assay. The affinity, but not the number, of [3H]N-methylscopolamine [( 3H]MS) binding sites was increased in the presence of diethyl ether (KD in air = 0.41 nM, KD in 2% diethyl ether = 0.21 nM). This increase in affinity reflected a decrease in the rapid dissociation rate constant (air k-1 = 13 X 10(-3) min-1, 2% diethyl ether k-1 = 7 X 10(-4) min-1) rather than a change in the association rate constant. Diethyl ether had no effect on the binding affinity of the muscarinic agonist carbamylcholine. However, the binding of a radiolabeled muscarinic agonist, [3H]oxotremorine-M [( 3H]Oxo-M), to high affinity binding sites decreased about 25% in the presence of 2% diethyl ether. The ability of a guanine nucleotide to depress the high affinity binding of both carbamylcholine and [3H]Oxo-M was decreased or eliminated by diethyl ether. Diethyl ether appears to interfere with muscarinic receptor-G protein interactions, perhaps by stabilizing receptor-G protein complexes or inhibiting the binding of guanine nucleotides.

Halothane attenuation of muscarinic inhibition of adenylate cyclase in rat heart.

Halothane stimulated basal adenylate cyclase activity in rat cardiac membranes. Maximal stimulation (54%) was obtained after equilibrating the membranes with 2% halothane. Halothane did not affect the fractional stimulation of adenylate cyclase activity produced by either forskolin or isoproterenol. However, halothane decreased carbamylcholine inhibition of adenylate cyclase activity stimulated by both forskolin and isoproterenol. Maximal depression of carbamylcholine inhibition of stimulated cyclase activity was obtained after equilibration with 1% halothane. These results are consistent with evidence from ligand binding studies and indicate that halothane disrupts muscarinic receptor-G-protein interactions.

Effects of halothane on high affinity agonist binding and guanine nucleotide sensitivity of muscarinic acetylcholine receptors from brainstem of rat.

The influence of halothane on muscarinic receptors with a high affinity for agonists was studied using [3H]oxotremorine-M. [3H]Oxotremorine-M bound with high affinity (KD = 2.8 nM) to a subpopulation of muscarinic receptors in the brainstem of rat, representing 32% of the total receptor pool. Agonist affinity for binding sites for [3H]oxotremorine-M was not affected by a guanine nucleotide (5'-guanylylimidodidiphosphate; Gpp(NH)p), although the level of binding was decreased, presumably due to the conversion of receptors to lower affinity conformations. However, only 58% of 3 nM binding of [3H]oxotremorine-M was sensitive to Gpp(NH)p. Halothane had two effects on the binding of [3H]oxotremorine-M: halothane (1) decreased the level of binding of [3H]oxotremorine-M without affecting agonist affinity for the surviving sites, and (2) lowered the sensitivity of the binding of [3H]oxotremorine-M to Gpp(NH)p by a factor of 120. The decrease in binding of [3H]oxotremorine-M binding was nonselective with regard to the sensitivity of the receptors to the guanine nucleotide, insofar as Gpp(NH)p inhibited the binding of [3H]oxotremorine-M to the same extent in the presence and absence of halothane. These results suggest that halothane (1) converts both G protein-coupled and -uncoupled muscarinic receptors to states of lower agonist affinity and (2) lowers the affinity of receptor-G protein complexes for guanine nucleotides.

Halothane effects on muscarinic acetylcholine receptor complexes in rat brain.

Muscarinic acetylcholine receptors in membranes from rat cerebral cortex or brainstem were equilibrated with halothane (0.5 to 5%). Halothane did not affect the number of [3H]methylscopolamine [( 3H]MS) binding sites. [3H]MS binding affinity, however, was increased in the presence of halothane (KD, air = 0.41 nM; KD, 2% halothane = 0.26 nM). This increase reflected a decrease in the dissociation rate constant (from 13 X 10(-3) min-1 to 6.5 X 10(-3) min-1) rather than a change in the bimolecular rate constant of association (1.8 and 1.9 X 10(7) M-1 min-1 in the absence and presence of 2% halothane respectively). Carbamylcholine affinity for brainstem or cortical muscarinic receptors was not affected by halothane. The ability of a guanine nucleotide to lower carbamylcholine affinity for brainstem receptors, however, was eliminated after equilibration with 2% halothane.

Biphasic regulation of detergent-solubilized muscarinic acetylcholine receptors from rat cerebral cortex by N-ethylmaleimide.

Muscarinic acetylcholine receptors were solubilized from rat cerebral cortex with 1% digitonin. Treatment with N-ethylmaleimide decreased or increased agonist affinity for the receptor, depending on the extent of alkylation. This indicates that certain determinants of receptor state do not depend on interactions with other membrane structures.