Adam C. Hall

Effects of inhalational general anaesthetics on native glycine receptors in rat medullary neurones and recombinant glycine receptors in Xenopus oocytes.

1 Glycine responses were studied under voltage clamp in Xenopus oocytes injected with cDNA encoding mammalian glycine receptor subunits and in rat medullary neurones. Bath application of glycine gave strychnine‐sensitive currents which reversed close to the expected equilibrium potentials for chloride ions. The peak currents for the receptors expressed in oocytes fitted a Hill equation with EC50 = 215 ± 5 μm and Hill coefficient nH = 1.70 ± 0.05 (means ± s.e.means). The peak currents from the receptors in medullary neurones fitted a Hill equation with EC50 = 30 ± 1 μm and Hill coefficient nH = 1.76 ± 0.08. The current‐voltage relationship for the receptors expressed in oocytes showed strong outward rectification (with Vrev = −21 ± 2 mV), while that for the glycine responses from the medullary neurones in symmetrical Cl− was linear (with Vrev = 3.2 ± 0.6 mV). 2 Inhalational general anaesthetics, at concentrations close to their human minimum alveolar concentrations (MACs), potentiated responses to low concentrations of glycine. The potentiation observed with the recombinant receptors (between 60–220%) was approximately twice that found with the medullary neurones (between 40–80%). For both the recombinant receptors and the receptors in medullary neurones, the degree of potentiation increased in the order of methoxyflurane ≅ sevoflurane < halothane ≅ isoflurane ≅ enflurane. There was no significant difference between the potentiations observed for the two optical isomers of isoflurane. 3 For both the recombinant and native receptors, isoflurane potentiated the currents in a dose‐dependent manner at low concentrations of glycine, although at high glycine concentrations the anaesthetic had no significant effect on the glycine‐activated responses. The major effect of isoflurane was to cause a parallel leftward shift in the glycine concentration‐response curves. The glycine EC50 concentration for the recombinant receptors decreased from a control value of 215 ± 5 μm to 84 ± 7 μm glycine at 610 μm isoflurane, while that for the medullary neurones decreased from a control value of 30 ± 1 μm to 18 ± 2 μm glycine at the same concentration of isoflurane. The potentiation was independent of membrane potential. 4 Isoflurane also potentiated responses to taurine, a partial agonist at the glycine receptor. This was observed for receptors expressed in oocytes at both low and saturating concentrations of taurine. The EC50 concentration decreased from a control value of 1.6 ± 0.2 to 0.9 ± 0.1 mM taurine in the presence of 305 μm isoflurane, while the maximum response to taurine increased from 47 ± 2 to 59 ± 2% of the maximum response to glycine. 5 Glycine receptors, like other members of the fast ligand‐gated receptor superfamily, are sensitive to clinically relevant concentrations of inhalational general anaesthetics. Effects at these receptors may, therefore, play some role in the maintenance of the anaesthetic state.

Stereoselective and non-stereoselective actions of isoflurane on the GABAA receptor.

1 Acutely dissociated cerebellar Purkinje neurones from 8–14 day old rats were studied under voltage clamp in the whole‐cell patch‐clamp configuration. Cl− currents induced by bath application of γ‐aminobutyric acid (GABA) were measured (using symmetrical Cl− solutions) at both low (2 μm) non‐desensitizing and high (300 μm) desensitizing concentrations of GABA. 2 At 2 μm GABA, the bicuculline‐sensitive Cl− currents were potentiated by racemic isoflurane and both of its optical isomers. Isoflurane had no effect on membrane current in the absence of GABA. The dose‐response data for potentiation by racemic isoflurane could be fitted with a Hill equation with an EC50 = 320 ± 20 μm isoflurane and a Hill coefficient of h = 2.7 ± 0.4 (means ± s.e.mean). 3 The potentiations produced by the optical isomers of isoflurane at 2 μm GABA were stereoselective at moderate and high anaesthetic concentrations. The maximum stereoselectivity, about two fold, occurred at the EC50 concentration for general anaesthesia (310 μm isoflurane), with S(+)‐isoflurane being more effective than R(−)‐isoflurane. At sub‐anaesthetic concentrations, the stereoselectivity was less marked and vanished at the lowest concentration used (77 μm isoflurane). 4 The sustained residual current remaining after exposure of neurones to a desensitizing concentration of GABA (300 μm) was inhibited non‐stereoselectively, but only at high concentrations of isoflurane. The ratio of inhibitions by S(+)‐ and R(−)‐isoflurane (mean ± s.e.mean) was 1.14 ± 0.21 at 770 μm isoflurane. At the EC50 concentration for general anaesthesia, however, the inhibition was barely significant. 5 The above results are discussed in relation to the possible role of the GABAA receptor channel in general anaesthesia.

Menthol shares general anesthetic activity and sites of action on the GABAA receptor with the intravenous agent, propofol

Menthol and related compounds were investigated for modulation of recombinant human gamma-aminobutyric acid type A (GABA(A), alpha(1)beta(2)gamma(2s)) receptor currents expressed in Xenopus oocytes. Sub-maximal (EC(20)) GABA currents were typically enhanced by co-applications of 3-300 microM (+)-menthol (e.g. by approximately 2-fold at 50 microM) > isopulegol > isomenthol> alpha-terpineol >> cyclohexanol. We studied menthols actions on GABA(A) receptors compared to sedatives (benzodiazepines) and intravenous anesthetics (barbiturates, steroids, etomidate and propofol). Flumazenil (a benzodiazepine antagonist) did not inhibit menthol enhancements while currents directly activated by 50 microM propofol were significantly inhibited (by 26+/-3%) by 50 microM (+)-menthol. GABA(A) receptors containing beta(2) subunits with either a point mutation in a methionine residue to a tryptophan at the 286 position (in transmembrane domain 3, TM-3) or a tyrosine to a tryptophan at the 444 position (TM-4) are insensitive to modulation by propofol. Enhancements of GABA EC(20) currents by menthol were equally abolished in GABA(A) alpha(1)beta(2)(M286W)gamma(2s) and alpha(1)beta(2)(Y444W)gamma(2s) receptors while positive modulations by benzodiazepines, barbiturates and steroids were unaffected. Menthol may therefore exert its actions on GABA(A) receptors via sites distinct from benzodiazepines, steroids and barbiturates, and via sites important for modulation by propofol. Finally, using an in vivo tadpole assay, addition of (+)-menthol resulted in a loss of righting reflex with an EC(50) of 23.5+/-4.7 microM (approximately10-fold less potent anesthesia than propofol). Thus, menthol and analogs share general anesthetic action with propofol, possibly via action at similar sites on the GABA(A) receptor.

Insensitivity of P- Type Calcium Channels to Inhalational and Intravenous General Anesthetics

BackgroundVoltage-gated Ca2+ channels long have been considered plausible targets for general anesthetics. Previous anesthetic studies have focused on L-, T-, or N-type channels, but there have been no studies on channels identified as P-type. Since P-type channels may be the most important voltage-gated Ca2+ channels involved in synaptic transmission in mammalian brain, it is important to establish their sensitivity to clinically relevant concentrations of general anesthetics. MethodsAcutely dissociated cerebellar Purkinje neurons were obtained from 7–14-day-old Sprague-Dawley rats. P-type currents were measured using the whole-cell version of the patch-clamp technique, with Ba2+ as the current carrier. General anesthetics were applied to the neurons in aqueous solution at room temperature (20–23°C). ResultsP-type Ca2+ channels were found to be very insensitive to a variety of general anesthetics and ethanol. Inhibitions of less than 10% were produced by 0.35 mM halothane, 0.35 μM isoflurane, 32 μM thiopental, 50 μM pentobarbital, 2 μM propofol, and 200 μM ethanol. Substantial anesthetic inhibition was found only at free aqueous concentrations much greater than those that are clinically relevant. For halothane, the dose-response curve showed an IC50 concentration of 1.17 ± 0.02 mM and a Hill coefficient of 2.02 ± 0.04 (mean ± SEM). ConclusionsThe relatively small Inhibitions of P-type Ca2+ channels produced by volatile and Intravenous anesthetics at their free aqueous EC50 concentrations for general anesthesia in mammals suggest that these channels do not play a major role in the induction of general anesthesia.

The relative potencies of dendrotoxins as blockers of the cloned voltage-gated K+ channel, mKv1.1 (MK-1), when stably expressed in Chinese hamster ovary cells

The mKv1.1 voltage‐gated K+ channel has been expressed stably in Chinese hamster ovary cells and whole‐cell currents recorded by the patch‐clamp method. A range of structurally related peptide toxins (dendrotoxins) from the venom of green mamba (Dendroaspis angusticeps) and black mamba (Dendroaspis polylepis polylepis) snakes were tested for mKv1.1 channel blocking activity. Their potencies were compared based on EC50s derived from their respective concentration‐inhibition relationships. The rank order of potency, thus determined was: Toxin K>γ‐dendrotoxin(γ‐Dtx)>δ‐Dtx>Toxin I=α‐Dtx>β‐Dtx. Block was independent of voltage and no effects of the toxins on the kinetics of activation were observed. These results are consistent with a mechanism involving the block of closed channels. A wide range of activity was observed even between toxins with an extremely high degree of sequence homology. Toxin K, in particular was an exquisitely potent blocker of the mKv1.1 channel, having an EC50 of 30 pm compared with 1.8 nm for δ‐Dtx in spite of 95% sequence identity.

Suprachiasmatic Nucleus Neurons Are Glucose Sensitive

The suprachiasmatic nucleus (SCN) in the hypothalamus serves as the pacemaker for mammalian circadian rhythms. In a hamster brain slice preparation, the authors were able to record spontaneous activity from SCN cells for up to 4 days in vitro and verify a self-sustained rhythm in firing. The phase of this rhythm was altered by the concentration of glucose in the bathing medium, with time of peak firing advanced for a 20 mM glucose condition and slightly delayed for a 5 mM glucose condition, relative to 10 mM. The advancing effect of 20 mM glucose and the delaying effect of 5 mM glucose were not maintained during a 2nd day in vitro after changing the bathing medium back to 10 mM glucose, thus indicating the effect was not a permanent phase shift of the underlying oscillation. In experiments recording from cell-attached membrane patches on acutely dissociated hamster SCN neurons, exchanging the bathing medium from high (20 mM) to zero glucose increased potassium (K+)-selective channel activity With inside-out membrane patches, the authors revealed the presence of a glybenclamide-sensitive K+ channel (190 pS) and a larger conductance (260 pS) Ca2+- dependent K+ channel that were both reversibly inhibited by ATP at the cytoplasmic surface. Furthermore, 1 mM tetraethylammonium chloride was demonstrated to advance peak firing time in the brain slice in a similar manner to a high concentration of glucose (20 mM). The authors interpret the results to imply that SCNs are sensitive to glucose, most probably via ATP modulation of K+ channel activity in these neurons. Tonic modulation of K+ channel activity appears to alter output of the pacemaker but does not reset the phase.

Blockade by dendrotoxin homologues of voltage-dependent K+ currents in cultured sensory neurones from neonatal rats.

1 Homologues of dendrotoxin (Dtx) were isolated from the crude venom of Green and Black Mamba snakes and examined for K+ channel blocking activity in neonatal rat dorsal root ganglion cells (DRGs) by whole‐cell patch clamp recording. 2 Outward potassium current activated by depolarization was composed of two major components: a slowly inactivating current (SIC, τdecay ≅ 50ms, 200 ms and 2 s), and a non‐inactivating current (NIC, τdecay > 2 min). Tail current analysis revealed two time constants of deactivation of total outward current, 3–12 ms and 50–150 ms (at −80 mV) which corresponded to SIC and NIC, respectively. 3 All the homologues (α‐, β‐, γ‐ and γ‐Dtx and toxins I and K) blocked outward current activated by depolarization in a dose‐dependent manner. The most potent in blocking total outward current was γ‐Dtx (EC50 of 0.5 ± 0.2 nm), although there were no statistically significant differences in potency between any of the homologues. 4 Qualitative differences in the nature of the block were noted between homologues. In particular, the block by γ‐Dtx was time‐dependent, whereas that by γ‐Dtx was not. 5 α‐Dtx was a much better blocker of SIC (EC50 = 1.0 ± 0.4 nm) than was γ‐Dtx (EC50 = 17.6 ± 5.8 nm). Furthermore, γ‐Dtx was selective for NIC (EC50 ± 0.24 ± 0.03 nm) over SIC and reduced the slow component of tail currents (NIC), preferentially. On the other hand, α‐Dtx did not significantly distinguish between SIC and NIC although tail current analysis showed that α‐Dtx preferentially reduced the fast component of tail currents (SIC). 6 The results confirm, using direct electrophysiological methods, that homologues of dendrotoxins from Mamba snake venom block K+ channels in rat sensory neurones. Furthermore, α‐Dtx and γ‐Dtx distinguish between sub‐types of K+ channels in these cells and may thus be useful pharmacological tools in other neuronal K+ channel studies.

Circadian modulation of the ryanodine receptor type 2 in the SCN of rodents.

We examined the temporal modulation of intracellular calcium release channels in the suprachiasmatic nucleus (SCN). We found a circadian rhythm in [3H]ryanodine binding that was specific to the SCN. The peak in the rhythm occurred at CT 7 and was due to an increase in Bmax, which correlated well with immunoblots showing an increase in RyR-2 expression in the SCN. Double immunohistochemical studies showed that RyR-2 was expressed exclusively in neurons. Ryanodine and caffeine applied around CT 7-9 advanced the clock phase in a hamster brain slice preparation. No rhythm of IP3R was seen in any of the brain areas studied. Our results indicate that RyR-2 exhibits an endogenous rhythm, which influences the intracellular calcium dynamics and thus modulates SCN activity.

Role of membrane conductances and protein synthesis in subjective day phase advances of the hamster circadian clock by neuropeptide Y.

Neurons of the mammalian circadian pacemaker in the hypothalamic suprachiasmatic nuclei exhibit a rhythm in firing rate that can be reset by neuropeptide Y. We recorded the effects of neuropeptide Y on Na+ and K+ conductances of hamster suprachiasmatic nuclei neurons using whole‐cell, perforated‐patch and cell‐attached patch‐clamp recordings, both in dissociated and brain slice preparations. While neuropeptide Y had no effect on voltage‐gated Na+ currents, neuropeptide Y activated a leak K+ current. Neuropeptide Y phase advances in the suprachiasmatic nuclei brain slice preparation were blocked by a number of K+ channel blockers (tetraethylammonium chloride, dendrotoxin‐I, glybenclamide). However, a K+ ionophore, valinomycin, did not shift the rhythm. The inhibition by tetraethylammonium chloride did not persist in the presence of glutamatergic receptor blockers. We have previously shown that glutamate can oppose neuropeptide Y phase‐shifting actions, suggesting that K+ channel inhibition acts by inducing glutamate release. Protein synthesis inhibitors had no effect on clock phase when applied during the subjective day, and had no influence on neuropeptide Y‐induced phase shifts. On the other hand, glutamates ability to inhibit neuropeptide Y shifts was abolished by protein synthesis inhibition. Thus, while neuropeptide Y phase shifts do not require protein synthesis, glutamate blocks neuropeptide Y shifts via increased gene expression during the subjective day, at a time when it does not reset the clock. These results indicate that neuropeptide Y phase shifts via a mechanism that does not involve changes in membrane conductance or protein synthesis.

Histamine Phase Shifts the Hamster Circadian Pacemaker via an NMDA Dependent Mechanism

The SCN acts as the central pacemaker for circadian rhythms in mammals. Histamine has been shown to affect circadian rhythms both in vivo and in vitro. We investigated the mechanism by which histamine phase shifts circadian rhythms in vitro. Hypothalamic slices containing the SCN were prepared from golden hamsters, and spontaneous firing rates of individual cells were recorded on the second day in vitro. Application of histamine (1 μM – 10 mM) at the extrapolated time of 2 h after lights off (ZT 14) on day 1 in vitro delayed the time of peak firing in a dose-dependent manner. Pre-exposure to the N-methyl-D-aspartate (NMDA) receptor antagonist (±)-2-amino-5-phosphonopentanoic acid (AP-5; 100 μM – 1mM) 5 min before histamine (1 μM) was applied to the slice blocked the phase-delaying effects of histamine. Application of the H1 blocker mepryamine (100 nM) or the H2 blocker cimetidine (10 μM) followed by histamine had no effect on the phase delay induced by histamine. In whole cell recordings from acutely dissociated neurons of hamster SCN, histamine (50 [.mu]M) was shown to potentiate NMDA-evoked currents by 52 ± 12%. These experiments demonstrate that histamine phase shifts of the circadian clock are dependent on NMDA receptor activation and that histamine can directly potentiate NMDA currents in SCN neurons. Histamine may alter circadian clock function by acting directly on NMDA receptors, possibly via binding to the polyamine site.