Henry U. Weigt

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 mmHg via the Langendorff technique initially with a salt solution at 37°C. Hearts were then perfused with fresh filtered (40-&mgr;m 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.

Atomoxetine acts as an NMDA receptor blocker in clinically relevant concentrations

Background and purpose:  There is increasing evidence that not only the monoaminergic but also the glutamatergic system is involved in the pathophysiology of attention‐deficit hyperactivity disorder (ADHD). Hyperactivity of glutamate metabolism might be causally related to a hypoactive state in the dopaminergic system. Atomoxetine, a selective noradrenaline reuptake inhibitor, is the first non‐stimulant approved for the treatment of this disorder. Here we have evaluated the effects of atomoxetine on glutamate receptors in vitro.

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. (Anesth Analg 1997;84:285-93)

Rapid increase of glial glutamate uptake via blockade of the protein kinase A pathway

Glutamate is the main excitatory neurotransmitter in the vertebrate central nervous system. Removal of this transmitter from the synaptic cleft by glial and neuronal transporter systems plays an important role in terminating glutamatergicneurotransmission. The effects of different activators and blockers of PKA and PKC on glutamate uptake were studied in primary glial cells cultivated from the rat cortex using the patch‐clamp recording technique and immunocytochemical methods. GF 109203X enhances glutamate‐induced membrane currents in a concentration‐ and time‐dependent manner. After pre‐application for 40 s the maximal transport capacity was increased by 30–80%. The estimated Km‐value of the transport system did not change after drug application and the enhanced glutamate uptake was reversible within a few minutes upon washout. Activators and blockers of the PKC pathway did not affect glutamate uptake, whereas H89, a selective blocker of PKA, mimicked the effects of GF 109203X, indicating involvement of the protein kinase A pathway. The GF 109203X‐induced increase in transport capacity is likely to be mediated by GLAST since the GLT‐1 selective blocker dihydrokainate was unable to block basal or stimulated glutamate uptake. Furthermore, the increase in transport activity may well be based on an increase in cell surface expression of the transporter protein since preincubation with cytochalasin‐B, a protein that blocks actin polymerization, almost completely abolished the effect of GF 109203X and H89. These results indicate that GF 109203X and H89 enhance glial glutamate uptake via blockade of the PKA. The described effect may affect glutamatergic neurotransmission by reducing the glutamate concentration in the synaptic cleft.

Modulation of the Cardiac Sodium Current by Inhalational Anesthetics in the Absence and Presence of β-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 sup + 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 sup + 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 micro Meter) was potentiated, further decreasing peak I sub Na by 34.7 +/‐ 4.1%. The halothane effect was less, although significant, in the presence of a G‐protein inhibitor (GDP beta S) or a specific protein kinase A inhibitor [PKI‐(6–22)‐amide], reducing peak I sub Na 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%. GDP beta S 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 sup + 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.

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

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.

Activation of Neuronal N-Methyl-D-Aspartate Receptor Channels by Lipid Emulsions

UNLABELLED Lipid emulsions are widely used as carriers for hypnotics such as propofol, etomidate, and diazepam. It is assumed that the emulsions alone exert no effect on cellular functions nor influence the pharmacokinetics, pharmacodynamics, or anesthetic and analgetic potency of the hypnotics they carry. To elucidate possible interactions between lipid emulsions and cell membranes, in particular membrane-bound proteins, we investigated the effects of commercially available lipid emulsions on the cell membranes of cultured cortical neurons from the mouse by using the whole-cell configuration of the patch-clamp technique. Of nine lipid emulsions tested, three, i.e., Intralipid, Structolipid, and, to a much lesser extent, Abbolipid, activated membrane currents in the neuronal cells in a dilution-dependent manner. The emulsion-induced currents were not affected by picrotoxin or bicuculline but were inhibited by DL-AP5 and ketamine. The voltage dependence of the currents was influenced by the presence of Mg(2+) in a way that is typical for currents conducted by N-methyl-D-aspartate receptor channels. We conclude that Intralipid, Structolipid, and Abbolipid activate N-methyl-D-aspartate receptor channels in cortical neurons. IMPLICATIONS Lipid emulsions are widely used as carriers for hypnotics such as propofol, etomidate, or diazepam. We tested nine commercially available lipid emulsions and demonstrate that three of them--Intralipid, Structolipid, and Abbolipid--activate NMDA receptor channels in the membranes of cortical neuronal cells.

Conformational State-dependent Effects of Halothane on Cardiac Na sup + Current

Background: The Na sup + channel is voltage gated and characterized by three distinct states: closed, open, and inactivated. To identify the effects of halothane on the cardiac Na sup + current (INa) at various membrane potentials, the effects of 1.2 mm halothane at different holding potentials (VH) on INa were examined in single, enzymatically isolated guinea pig ventricular myocytes. Methods: The INa was recorded using the whole‐cell configuration of the patch‐clamp technique. Currents were generated from resting VH 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) VH ‐dependent depressions of peak INa (mean +/‐ SEM): 24.4 +/‐ 4.1% (VH = ‐110 mV), 42.1 +/‐ 3.4% (VH = ‐80 mV), and 75.2 +/‐ 1.5% (VH = ‐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 VH of ‐80 mV, halothane exhibited a use‐dependent block, with block of INa 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 VH of ‐110 mV. When inactivation of INa was removed by exposure to 100 micro Meter veratridine, no significant difference was observed in the depressant effect of halothane at both VH s: 26.6 +/‐ 4.5% (VH = ‐80 mV) and 26.4 +/‐ 5.6% (VH = ‐110 mV). Conclusions: The present findings indicate that the depressant action of halothane on cardiac INa 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 VH, but depression of the current was still evident. These results indicate a complex interaction between halothane and the various conformational states of the Na sup + channel.

Modulation of Cardiac Sodium Current by α1-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 sup + current (INa). 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 INa in the absence and presence of equianesthetic concentrations of halothane and isoflurane in single ventricular myocytes from adult guinea pig hearts. Methods: INa was recorded using the standard whole‐cell configuration of the patch‐clamp technique. Voltage clamp protocols initiated from two different holding potentials (VH) 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 INa in a concentration‐ and voltage‐dependent manner, being more potent at the depolarized VH. Halothane and isoflurane interacted synergistically with methoxamine to suppress INa near the physiologic cardiac resting potential of ‐80 mV. The effect of methoxamine with anesthetics appeared to be additive when using a VH of ‐110 mV, a potential where no Na sup + 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 sup + 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 sup + channels in the inactivated state.

Evidence That Xenon Does Not Produce Open Channel Blockade of the NMDA Receptor

Previous studies had not excluded the possibility that the mechanism by which Xenon (Xe) blocks N-methyl-D-aspartate (NMDA) receptors might be that of an open-channel blocker. We tested this possibility on mutant NMDA receptors carrying an alanine (A) to cysteine (C) mutation located within the SYTANLAAF-motif of the third transmembrane region (TM3). This mutation was shown to yield constitutively open ion channels after modification with a thiol-modifying reagent. We expressed such mutant channels in Neuro2A cells and recorded glutamate (50 microM)-induced currents in the whole cell recording mode. Although Xe (3.5 mM) blocked the currents through the wild-type receptor NR1-1a/NR2A and NR1-1a/NR2B by approximately 40% and those through the mutant receptors NR1-1a/NR2A(A650C) or NR1-1a/NR2B(A651C) by approximately 30%, it was unable to block the currents through the methane thiosulfonate etyhlammonium-modified mutant receptors. On the other hand, established open-channel blockers of the NMDA receptor such as MK-801 (1 microM) or Mg ions (Mg(2+); 1 mM) were able to block these permanently open channels. These results suggest that Xe does not act as a classical open-channel blocker at the NMDA receptor.