Intranasal application of xenon: describing the pharmacokinetics in experimental animals and the increased pain tolerance within a placebo-controlled experimental human study.

BACKGROUND: Pain sensitizes the central nervous system via N-methyl-D-aspartate receptors (NMDARs) leading to an enhancement of pain perception. However, the enhanced responsiveness of pain-processing areas can be suppressed by subanaesthetic doses of the NMDAR antagonist xenon. To analyse the strength of the analgesic effect of low-dose xenon using new economical application methods, we tested xenon applied nasally in an experimental human pain setting. METHODS: We tested 10 healthy volunteers using a multimodal experimental pain testing in a randomized double-blind placebo-controlled repeated measures study. Xenon was administered using a novel low-pressure intranasal application device. Additionally, we measured xenon concentrations in blood samples obtained from intracranial veins of experimental animals to describe the pharmacokinetics of intranasally applied xenon in the cerebral compartment. RESULTS: Intranasal application of xenon at a rate of 1.0 litre h(-1) for 30 min significantly increased pain tolerance of volunteers to ischaemic (+128%), cold (+58%), and mechanical (+40%) stimulation (P<0.01). However, 60 min after terminating the application of xenon, there was no significant alteration of pain tolerance compared with placebo. Cranial blood concentrations of xenon in pigs reached a steady state of approximately 450 nl ml(-1) after 5 min. CONCLUSIONS: In this placebo-controlled experimental human study, we described the increased pain tolerance induced by intranasally applied xenon. On the basis of our results, we conclude that intranasally administered xenon has analgesic properties and suggest that the novel application device presented here offers new possibilities for the administration of NMDAR antagonists within a multimodal analgesia approach.

Etomidate reduces glutamate uptake in rat cultured glial cells: involvement of PKA.

BACKGROUND AND PURPOSE: Glutamate is the main excitatory neurotransmitter in the vertebrate CNS. Removal of the transmitter from the synaptic cleft by glial and neuronal glutamate transporters (GLTs) has an important function in terminating glutamatergic neurotransmission and neurological disorders. Five distinct excitatory amino-acid transporters have been characterized, among which the glial transporters excitatory amino-acid transporter 1 (EAAT1) (glutamate aspartate transporter) and EAAT2 (GLT1) are most important for the removal of extracellular glutamate. The purpose of this study was to describe the effect of the commonly used anaesthetic etomidate on glutamate uptake in cultures of glial cells. EXPERIMENTAL APPROACH: The activity of the transporters was determined electrophysiologically using the whole cell configuration of the patch-clamp recording technique. KEY RESULTS: Glutamate uptake was suppressed by etomidate (3-100 microM) in a time- and concentration-dependent manner with a half-maximum effect occurring at 2.4+/-0.6 microM. Maximum inhibition was approximately 50% with respect to the control. Etomidate led to a significant decrease of V(max) whereas the K(m) of the transporter was unaffected. In all cases, suppression of glutamate uptake was reversible within a few minutes upon washout. Furthermore, both GF 109203X, a nonselective inhibitor of PKs, and H89, a selective blocker of PKA, completely abolished the inhibitory effect of etomidate. CONCLUSION AND IMPLICATIONS: Inhibition of glutamate uptake by etomidate at clinically relevant concentrations may affect glutamatergic neurotransmission by increasing the glutamate concentration in the synaptic cleft and may compromise patients suffering from acute or chronic neurological disorders such as CNS trauma or epilepsy.

Xenon reduces glutamate-, AMPA-, and kainate-induced membrane currents in cortical neurones.

BACKGROUND: The anaesthetic, analgesic, and neuroprotective effects of xenon (Xe) are believed to be mediated by a block of the NMDA (N-methyl-D-aspartate) receptor channel. Interestingly, the clinical profile of the noble gas differs markedly from that of specific NMDA receptor antagonists. The aim of this study was, therefore, to investigate whether Xe might be less specific, also inhibiting the two other subtypes of glutamate receptor channels, such as the alpha-amino-3-hydroxy-5-methyl-4-isoxazolole propionate (AMPA) and kainate receptors. METHODS: The study was performed on voltage-clamped cortical neurones from embryonic mice and SH-SY5Y cells expressing GluR6 kainate receptors. Drugs were applied by a multi-barreled fast perfusion system. RESULTS: Xe, dissolved at approximately 3.45 mM in aqueous solution, diminished the peak and even more the plateau of AMPA and glutamate induced currents. At the control EC(50) value for AMPA (29 microM) these reductions were by about 40 and 56% and at 3 mM glutamate the reductions were by 45 and 66%, respectively. Currents activated at the control EC(50) value for kainate (57 microM) were inhibited by 42%. Likewise, Xe showed an inhibitory effect on kainate-induced membrane currents of SH-SY5Y cells transfected with the GluR6 subunit of the kainate receptor. Xe reduced kainate-induced currents by between 35 and 60%, depending on the kainate concentration. CONCLUSIONS: Xe blocks not only NMDA receptors, but also AMPA and kainate receptors in cortical neurones as well as GluR6-type receptors expressed in SH-SY5Y cells. Thus, Xe seems to be rather non-specific as a channel blocker and this may contribute to the analgesic and anaesthetic potency of Xe.

Xenon incorporated in a lipid emulsion inhibits NMDA receptor channels.

BACKGROUND: Over the past decade hyperpolarized (129)xenon incorporated in lipid emulsions has been studied for the purpose of imaging enhancement in radiology. Xenon (Xe), a NMDA (N-methyl-D-aspartate)-receptor antagonist, has neuroprotective properties even at subanesthetic concentrations. Thus, its intravenous administration for this purpose deserves further evaluation. In this study, we investigated in an in vitro model the effect of Xe, incorporated in a lipid emulsion (Lipofundin MCT(R) 20%), on the NMDA receptor channel of cortical neurons of the mouse. METHODS: Pulses of 50 micro M of NMDA solution were extracellularly applied to the cells for 10 s, and the elicited membrane currents (I) were recorded while the membrane potential (V) was clamped at -80 mV. Either Lipofundin MCT(R) 20% or aqueous solution was loaded with Xe and applied simultaneously with the NMDA pulses by means of a multibarreled pipette attached to a battery of infusion-pumps. RESULTS: Xenon equilibrated in Lipofundin(R) caused a concentration-dependent and reversible inhibition of NMDA-induced currents (maximal Xe content [Xemax]: 190 micro l ml-1). The inhibitory effect was equivalent compared with the effect of Xe dissolved in aqueous solution (Xemax: 89 micro l ml-1) even though the Xe content of the lipid solution was almost doubled. Further enhancement of the Xe content by saturating both the lipid emulsion and the aqueous solutions with Xe (Xemax: 256 micro l ml-1) did not increase the inhibitory action on NMDA-receptors. CONCLUSION: The data demonstrate that Xe dissolved in Lipofundin MCT(R) 20% inhibits NMDA-receptors. Lipid emulsions enriched with Xe may serve as a carrier and a reservoir for Xe.

Xenon does not alter cardiac function or major cation currents in isolated guinea pig hearts or myocytes.

BACKGROUND: The noble gas xenon (Xe) has been used as an inhalational anesthetic agent in clinical trials with little or no physiologic side effects. Like nitrous oxide, Xe is believed to exert minimal unwanted cardiovascular effects, and like nitrous oxide, the vapor concentration to achieve 1 minimum alveolar concentration (MAC) for Xe in humans is high, i.e., 70-80%. In the current study, concentrations of up to 80% Xe were examined for possible myocardial effects in isolated, erythrocyte-perfused guinea pig hearts and for possible effects on altering major cation currents in isolated guinea pig cardiomyocytes. METHODS: Isolated guinea pigs hearts were perfused at 70 mm Hg via the Langendorff technique initially with a salt solution at 37 degrees C. Hearts were then perfused with fresh filtered (40-microm pore) and washed canine erythrocytes diluted in the salt solution equilibrated with 20% O2 in nitrogen (control), with 20% O2, 40% Xe, and 40% N2, (0.5 MAC), or with 20% O2 and 80% Xe (1 MAC), respectively. Hearts were perfused with 80% Xe for 15 min, and bradykinin was injected into the blood perfusate to test endothelium-dependent vasodilatory responses. Using the whole-cell patch-clamp technique, 80% Xe was tested for effects on the cardiac ion currents, the Na+, the L-type Ca2+, and the inward-rectifier K+ channel, in guinea pig myocytes suffused with a salt solution equilibrated with the same combinations of Xe, oxygen, and nitrogen as above. RESULTS: In isolated hearts, heart rate, atrioventricular conduction time, left ventricular pressure, coronary flow, oxygen extraction, oxygen consumption, cardiac efficiency, and flow responses to bradykinin were not significantly (repeated measures analysis of variance, P>0.05) altered by 40% or 80% Xe compared with controls. In isolated cardiomyocytes, the amplitudes of the Na+, the L-type Ca2+, and the inward-rectifier K+ channel over a range of voltages also were not altered by 80% Xe compared with controls. CONCLUSIONS: Unlike hydrocarbon-based gaseous anesthetics, Xe does not significantly alter any measured electrical, mechanical, or metabolic factors, or the nitric oxide-dependent flow response in isolated hearts, at least partly because Xe does not alter the major cation currents as shown here for cardiac myocytes. The authors' results indicate that Xe, at approximately 1 MAC for humans, has no physiologically important effects on the guinea pig heart.

Modulation of the cardiac sodium current by inhalational anesthetics in the absence and presence of beta-stimulation.

BACKGROUND: Cardiac dysrhythmias during inhalational anesthesia in association with catecholamines are well known, and halothane is more "sensitizing" than isoflurane. However, the underlying mechanisms of action of volatile anesthetics with or without catecholamines on cardiac Na channels are poorly understood. In this study, the authors investigated the effects of halothane and isoflurane in the absence and presence of beta-stimulation (isoproterenol) on the cardiac Na+ current (INa) in ventricular myocytes enzymatically isolated from adult guinea pig hearts. METHODS: A standard whole-cell patch-clamp technique was used. The INa was elicited by depolarizing test pulses from a holding potential of -80 mV in reduced Na+ solution (10 mM). RESULTS: Isoproterenol alone depressed peak INa significantly by 14.6 +/- 1.7% (means +/- SEM). Halothane (1.2 mM) and isoflurane (1.0 mM) also depressed peak INa significantly by 42.1 +/- 3.4% and 21.3 +/- 1.9%, respectively. In the presence of halothane, the effect of isoproterenol (1 microM) was potentiated, further decreasing peak INa by 34.7 +/- 4.1%. The halothane effect was less, although significant, in the presence of a G-protein inhibitor (GDPbetaS) or a specific protein kinase A inhibitor [PKI-(6-22)-amide], reducing peak INa by 24.2 +/- 3.3% and 24 +/- 2.4%, respectively. In combination with isoflurane, the effect of isoproterenol on INa inhibition was less pronounced, but significant, decreasing current by 12.6 +/- 3.9%. GDPbetaS also reduced the inhibitory effect of isoflurane. In contrast, PKI-(6-22)-amide had no effect on isoflurane INa inhibition. CONCLUSIONS: These results suggest two distinct pathways for volatile anesthetic modulation on the cardiac Na+ current: (1) involvement of G proteins and a cyclic adenosine monophosphate (cAMP)-mediated pathway for halothane and, (2) a G-protein-dependent but cAMP-independent pathway for isoflurane. Furthermore, these studies show that the inhibition of cardiac INa by isoproterenol is enhanced in the presence of halothane, suggesting some form of synergistic interaction between halothane and isoproterenol.

Sensitization of the cardiac Na channel to alpha1-adrenergic stimulation by inhalation anesthetics: evidence for distinct modulatory pathways.

BACKGROUND: Alpha1-adrenergic receptor stimulation has been shown to inhibit cardiac Na+ current (INa). Furthermore, some form of synergistic interaction of alpha1-adrenergic effects on INa in combination with volatile anesthetics has been reported. In this study, the authors investigated the possible role of G proteins and protein kinase C in the effects of halothane and isoflurane in the absence and presence of alpha1-adrenergic stimulation on the cardiac INa. METHODS: The standard whole-cell configuration of the patch-clamp technique was used. INa was elicited by depolarizing test pulses from a holding potential of -80 mV in reduced Na+ solution (10 mM). The experiments were conducted on ventricular myocytes enzymatically isolated from adult guinea pig hearts. RESULTS: The inhibitory effect of halothane (1.2 mM) and isoflurane (1 mM) on peak INa was significantly diminished in the presence of guanosine 5'-O-[2-thiodiphosphate (GDPbetaS). In myocytes pretreated with pertussis toxin (PTX), the potency of halothane was significantly enhanced, but the isoflurane effect was unchanged. In the presence of the protein kinase C (PKC) inhibitor bisindolylmaleimide (BIS), the effect of halothane was unchanged. In contrast, the effect of isoflurane on INa in the presence of BIS was significantly enhanced. The positive interaction between methoxamine and halothane was evident in the presence of G protein and PKC inhibitors. In contrast, the effect of methoxamine with isoflurane was additive in the presence of GDPbetaS or BIS. CONCLUSIONS: Different second messenger systems are involved in the regulation of cardiac Na+ current by volatile anesthetics. The effect of halothane involves a complex interaction with G proteins but is independent of regulation by PKC. In contrast, PKC is involved in the modulation of cardiac INa by isoflurane. In addition, non-PTX-sensitive G proteins may contribute to the effects of isoflurane. The positive interaction between methoxamine and anesthetics are independent of G proteins and PKC for halothane. In the case of isoflurane, the positive interaction with methoxamine is coupled to PTX-insensitive G proteins and PKC.

Voltage-dependent effects of volatile anesthetics on cardiac sodium current.

Cardiac dysrhythmias during inhaled anesthesia are well documented and may, in part, involve depression of the fast inward Na+ current (INa) during the action potential upstroke. In this study, we examined the effects of halothane, isoflurane, and sevoflurane at clinically relevant concentrations on INa in single ventricular myocytes isolated enzymatically from adult guinea pig hearts. INa was recorded using standard whole-cell configuration of the patch clamp technique. Halothane at 0.6 mM and 1.2 mM produced significant (P < 0.05) depressions of peak INa of 12.3% +/- 1.8% and 24.4% +/- 4.1% (mean +/- SEM, n = 12), respectively. Isoflurane (0.5 mM, n = 12; 1.0 mM, n = 15) and sevoflurane (0.6 mM, n = 14; 1.2 mM, n = 12) were less potent than halothane, decreasing peak INa by 4.8% +/- 1.1% and 11.4% +/- 1.4% (isoflurane) and 3.0% +/- 0.7% and 10.7% +/- 3.9% (sevoflurane). The depressant effects on INa were reversible in all cases. For all anesthetics tested, the degree of block increased at more depolarizing potentials. Anesthetics induced significant shifts in the steady-state inactivation and activation of the channel toward more hyperpolarizing potentials. The present findings indicate that volatile anesthetics at clinical concentrations decrease the cardiac INa in a dose- and voltage-dependent manner. At approximately equianesthetic concentrations, the decrease of INa caused by halothane was twice that observed with isoflurane or sevoflurane.

Conformational state-dependent effects of halothane on cardiac Na+ current.

BACKGROUND: The Na+ channel is voltage gated and characterized by three distinct states: closed, open, and inactivated. To identify the effects of halothane on the cardiac Na+ current (I(Na)) at various membrane potentials, the effects of 1.2 mM halothane at different holding potentials (V(H)) on I(Na) were examined in single, enzymatically isolated guinea pig ventricular myocytes. METHODS: The I(Na) was recorded using the whole-cell configuration of the patch-clamp technique. Currents were generated from resting V(H)s of -110, -80, or -65 mV. State-dependent block was characterized by monitoring frequency dependence, tonic block, and removal of inactivation by veratridine. RESULTS: Halothane produced significant (P < 0.05) V(H)-dependent depressions of peak I(Na) (mean +/- SEM): 24.4 +/- 4.1% (V(H) = -110 mV), 42.1 +/- 3.4% (V(H) = -80 mV), and 75.2 +/- 1.5% (V(H) = -65 mV). Recovery from inactivation was significantly increased when cells were held at -80 mV (control, tau = 6.0 +/- 0.3 ms; halothane, tau = 7.1 +/- 0.4 ms), but not at -110 mV. When using a V(H) of -80 mV, halothane exhibited a use-dependent block, with block of I(Na) increasing from 8.6 +/- 1.4% to 30.7 +/- 3.5% at test pulse rates of 2 and 11 Hz, respectively. Use-dependent inhibition was not apparent at V(H) of -110 mV. When inactivation of I(Na) was removed by exposure to 100 microM veratridine, no significant difference was observed in the depressant effect of halothane at both V(H)s: 26.6 +/- 4.5% (V(H) = -80 mV) and 26.4 +/- 5.6% (V(H) = -110 mV). CONCLUSIONS: The present findings indicate that the depressant action of halothane on cardiac I(Na) depends on the conformational state of the channel. As more channels are in the inactivated state, the more potent is the effect of halothane. Removal of channel inactivation by veratridine abolished the dependence of the halothane effect on V(H), but depression of the current was still evident. These results indicate a complex interaction between halothane and the various conformational states of the Na+ channel.

Modulation of cardiac sodium current by alpha1-stimulation and volatile anesthetics.

BACKGROUND: Alpha1-adrenoceptor stimulation is known to produce electrophysiologic changes in cardiac tissues, which may involve modulations of the fast inward Na+ current (I(Na)). A direct prodysrhythmic alpha1-mediated interaction between catecholamines and halothane has been demonstrated, supporting the hypothesis that generation of halothane-epinephrine dysrhythmias may involve slowed conduction, leading to reentry. In this study, we examined the effects of a selective alpha1-adrenergic receptor agonist, methoxamine, on cardiac I(Na) in the absence and presence of equianesthetic concentrations of halothane and isoflurane in single ventricular myocytes from adult guinea pig hearts. METHODS: I(Na) was recorded using the standard whole-cell configuration of the patch-clamp technique. Voltage clamp protocols initiated from two different holding potentials (V(H)) were applied to examine state-dependent effects of methoxamine in the presence of anesthetics. Steady state activation and inactivation and recovery from inactivation were characterized using standard protocols. RESULTS: Methoxamine decreased I(Na) in a concentration- and voltage-dependent manner, being more potent at the depolarized V(H). Halothane and isoflurane interacted synergistically with methoxamine to suppress I(Na) near the physiologic cardiac resting potential of -80 mV. The effect of methoxamine with anesthetics appeared to be additive when using a V(H) of -110 mV, a potential where no Na+ channels are in the inactivated state. Methoxamine in the absence and presence of anesthetics significantly shifted the half maximal inactivation voltage in the hyperpolarizing direction but had no effect on steady-state activation. CONCLUSION: The present results show that methoxamine (alpha1-adrenergic stimulation) decreases cardiac Na+ current in a concentration- and voltage-dependent manner. Further, a form of synergistic interaction between methoxamine and inhalational anesthetics, halothane and isoflurane, was observed. This interaction appears to depend on the fraction of Na+ channels in the inactivated state. (Key words: Anesthetics, volatile: halothane; isoflurane; methoxamine. Patch clamp: whole-cell configuration; sodium current; ventricular guinea pig myocytes.)