Wai-Meng Kwok

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.

Painful neuropathy decreases membrane calcium current in mammalian primary afferent neurons

&NA; Hyperexcitability of the primary afferent neuron leads to neuropathic pain following injury to peripheral axons. Changes in calcium channel function of sensory neurons following injury have not been directly examined at the channel level, even though calcium is a primary second messenger‐regulating neuronal function. We compared calcium currents (ICa) in 101 acutely isolated dorsal root ganglion neurons from 31 rats with neuropathic pain following chronic constriction injury (CCI) of the sciatic nerve, to cells from 25 rats with normal sensory function following sham surgery. Cells projecting to the sciatic nerve were identified with a fluorescent label applied at the CCI site. Membrane function was determined using patch‐clamp techniques in current clamp mode, and in voltage‐clamp mode using solutions and conditions designed to isolate ICa. Somata of peripheral sensory neurons from hyperalgesic rats demonstrated decreased ICa. Peak calcium channel current density was diminished by injury from 3.06±0.30 pS/pF to 2.22±0.26 pS/pF in medium neurons, and from 3.93±0.38 pS/pF to 2.99±0.40 pS/pF in large neurons. Under these voltage and pharmacologic conditions, medium‐sized neuropathic cells lacked obvious T‐type calcium currents which were present in 25% of medium‐sized cells from control animals. Altered Ca2+ signalling in injured sensory neurons may contribute to hyperexcitability leading to neuropathic pain.

Gabapentin decreases membrane calcium currents in injured as well as in control mammalian primary afferent neurons.

Background and Objectives Neuropathic pain following injury to peripheral sensory neurons is a common clinical problem and frequently difficult to treat. Gabapentin (GBP), a novel anticonvulsant, has significant analgesic effects in clinical neuropathic states and in relevant preclinical models, but its mechanism of action remains unclear. Because calcium currents play a significant role in neuronal function, this study was designed to assess the effect of GBP on the membrane voltage-activated inward calcium currents (ICa) in dorsal root ganglia (DRG) primary afferent neurons of neuropathic versus control rats. Methods Male rats were prepared according to the chronic constriction injury (CCI) model. The L4 and L5 dorsal root ganglia of those selected as CCI or control after appropriate behavioral testing were removed, and neurons were enzymatically dissociated. Fluorescent dye (DiI) placed at the injury site allowed identification of neurons projecting to that site. These were acutely studied using whole-cell, perforated (with β-escin) patch-clamp recordings. Additionally, neurons from sham or nonoperated rats were also studied. Results Although there was marked variability among cells, concentrations of GBP ranging from 0.1 to 300 μmol/L decreased neuronal peak ICa in midsized neurons (30 to 40 μm) of both sham and neuropathic rats, in a fast, reversible, and concentration-dependent manner. Intergroup differences were not significant, however the concentration-response EC50s were 2.7 μmol/L for the sham and 16.5 μmol/L for the CCI neurons. The drug suppressed ICa in nonoperated rats to a lesser degree, but changes did not differ significantly from the operated groups. Calcium currents in either small or large diameter neurons were also variably decreased by 10 μmol/L of GBP in sham and CCI neurons. Current inhibition by GBP was partly voltage dependent. Conclusions GBP, at clinically relevant concentrations, results in significant reduction of ICa in both sham and neuropathic neurons, while in nonoperated rats reduced ICa to a smaller degree. Sensitivity to drug was not affected by neuropathy. This current inhibition is partly voltage dependent. Depression of ICa may be partly related to the binding of the drug to the α2δ modulatory subunit of the voltage activated calcium channels (VACC). Analgesia may be due to diminished release of neurotransmitter by sensory neurons, a Ca2+-dependent process.

Nitric oxide activates ATP-sensitive potassium channels in mammalian sensory neurons: action by direct S-nitrosylation

BackgroundATP-sensitive potassium (KATP) channels in neurons regulate excitability, neurotransmitter release and mediate protection from cell-death. Furthermore, activation of KATP channels is suppressed in DRG neurons after painful-like nerve injury. NO-dependent mechanisms modulate both KATP channels and participate in the pathophysiology and pharmacology of neuropathic pain. Therefore, we investigated NO modulation of KATP channels in control and axotomized DRG neurons.ResultsCell-attached and cell-free recordings of KATP currents in large DRG neurons from control rats (sham surgery, SS) revealed activation of KATP channels by NO exogenously released by the NO donor SNAP, through decreased sensitivity to [ATP]i.This NO-induced KATP channel activation was not altered in ganglia from animals that demonstrated sustained hyperalgesia-type response to nociceptive stimulation following spinal nerve ligation. However, baseline opening of KATP channels and their activation induced by metabolic inhibition was suppressed by axotomy. Failure to block the NO-mediated amplification of KATP currents with specific inhibitors of sGC and PKG indicated that the classical sGC/cGMP/PKG signaling pathway was not involved in the activation by SNAP. NO-induced activation of KATP channels remained intact in cell-free patches, was reversed by DTT, a thiol-reducing agent, and prevented by NEM, a thiol-alkylating agent. Other findings indicated that the mechanisms by which NO activates KATP channels involve direct S-nitrosylation of cysteine residues in the SUR1 subunit. Specifically, current through recombinant wild-type SUR1/Kir6.2 channels expressed in COS7 cells was activated by NO, but channels formed only from truncated isoform Kir6.2 subunits without SUR1 subunits were insensitive to NO. Further, mutagenesis of SUR1 indicated that NO-induced KATP channel activation involves interaction of NO with residues in the NBD1 of the SUR1 subunit.ConclusionNO activates KATP channels in large DRG neurons via direct S-nitrosylation of cysteine residues in the SUR1 subunit. The capacity of NO to activate KATP channels via this mechanism remains intact even after spinal nerve ligation, thus providing opportunities for selective pharmacological enhancement of KATP current even after decrease of this current by painful-like nerve injury.

The A3 adenosine receptor agonist CP-532,903 [N6-(2,5-dichlorobenzyl)-3'-aminoadenosine-5'-N-methylcarboxamide] protects against myocardial ischemia/reperfusion injury via the sarcolemmal ATP-sensitive potassium channel.

We examined the cardioprotective profile of the new A3 adenosine receptor (AR) agonist CP-532,903 [N6-(2,5-dichlorobenzyl)-3′-aminoadenosine-5′-N-methylcarboxamide] in an in vivo mouse model of infarction and an isolated heart model of global ischemia/reperfusion injury. In radioligand binding and cAMP accumulation assays using human embryonic kidney 293 cells expressing recombinant mouse ARs, CP-532,903 was found to bind with high affinity to mouse A3ARs (Ki = 9.0 ± 2.5 nM) and with high selectivity versus mouse A1AR (100-fold) and A2AARs (1000-fold). In in vivo ischemia/reperfusion experiments, pretreating mice with 30 or 100 μg/kg CP-532,903 reduced infarct size from 59.2 ± 2.1% of the risk region in vehicle-treated mice to 42.5 ± 2.3 and 39.0 ± 2.9%, respectively. Likewise, treating isolated mouse hearts with CP-532,903 (10, 30, or 100 nM) concentration dependently improved recovery of contractile function after 20 min of global ischemia and 45 min of reperfusion, including developed pressure and maximal rate of contraction/relaxation. In both models of ischemia/reperfusion injury, CP-532,903 provided no benefit in studies using mice with genetic disruption of the A3AR gene, A3 knockout (KO) mice. In isolated heart studies, protection provided by CP-532,903 and ischemic preconditioning induced by three brief ischemia/reperfusion cycles were lost in Kir6.2 KO mice lacking expression of the pore-forming subunit of the sarcolemmal ATP-sensitive potassium (KATP) channel. Whole-cell patch-clamp recordings provided evidence that the A3AR is functionally coupled to the sarcolemmal KATP channel in murine cardiomyocytes. We conclude that CP-532,903 is a highly selective agonist of the mouse A3AR that protects against ischemia/reperfusion injury by activating sarcolemmal KATP channels.

Isoflurane-induced facilitation of the cardiac sarcolemmal KATP channel

Background Volatile anesthetics have cardioprotective effects that mimic ischemic preconditioning, including the involvement of adenosine triphosphate–sensitive potassium (KATP) channels. However, evidence for a direct effect of volatile anesthetic on the KATP channel is limited. In this study, the effects of isoflurane on the cardiac sarcolemmal KATP channel were investigated. Methods Single ventricular myocytes were enzymatically isolated from guinea pig hearts. Whole cell and single-channel configurations, specifically the cell-attached and inside-out patch mode, of the patch clamp technique were used to monitor sarcolemmal KATP channel current. Results In the cell-attached patch configuration, 2,4-dinitrophenol (150 &mgr;m) opened the sarcolemmal KATP channel. Isoflurane (0.5 mm) further increased channel open probability and the number of active channels in the patch. In contrast, in the inside-out patch experiments, isoflurane had no significant effect on the KATP channel activated by low ATP (0.2–0.5 mm). In addition, isoflurane had no effect on the KATP channel when activated by adenosine diphosphate, adenosine + guanosine triphosphate, bimakalim, and 2,4-dinitrophenol under inside-out patch configurations. When KATP current was monitored in the whole cell mode, isoflurane alone was unable to elicit channel opening. However, during sustained protein kinase C activation by 12,13-dibutyrate, isoflurane activated the KATP current that was sensitive to glibenclamide. In contrast, isoflurane had no effect on the KATP channel activated by 12,13-dibutyrate in a cell-free environment. Conclusions Isoflurane facilitated the opening of the sarcolemmal KATP channel in the intact cell, but not in an excised, inside-out patch. The isoflurane effect was not due to a direct interaction with the KATP channel protein, but required an intracellular component, likely including the translocation of specific protein kinase C isoforms. This suggests that the sarcolemmal KATP channel may have a significant role in anesthetic-induced preconditioning.

Loss of T-type calcium current in sensory neurons of rats with neuropathic pain.

Background Pathophysiology in the primary sensory neuron may contribute to chronic neuropathic pain. Ca channels play a central role in neuronal processes, and sensory neurons are rich in low-voltage–activated calcium channels (LVACCs). However, the physiologic function of these channels is unknown. Their possible role in rebound burst firing makes them a candidate for increased excitability after neuropathic injury. Methods This study uses pharmacological methods to isolate LVACC in cells from the dorsal root ganglia of neuropathic and sham-operated rats, including the blockade of high-voltage–activated Ca channels with fluoride and selective toxins. LVACCs were examined with conventional whole cell patch clamp electrophysiology techniques. Results After chronic constriction injury of the peripheral axon, LVACC was significantly reduced compared to sham rats as shown by a 60% reduction in peak current density and an 80% reduction in total calcium influx. A depolarizing shift in the voltage dependence of activation and an increase in the rate of deactivation and inactivation appear to cause this reduction of LVACC. Either Ni2+ or mibefradil, blockers of LVACC, applied in the bath to normal dorsal root ganglion cells during current clamp significantly and reversibly increased excitability. Conclusions These results suggest that loss of LVACC may contribute to decreased spike frequency adaptation and increased excitability after injury to sensory neurons. Through decreased Ca2+ influx, the cell becomes less stable and more likely to initiate or transmit bursts of action potentials. Consequently, modulation of Ca2+ currents at the dorsal root ganglion may be a potential method of therapeutic intervention.

Store-Operated Ca2+ Entry in Sensory Neurons: Functional Role and the Effect of Painful Nerve Injury

Painful nerve injury disrupts levels of cytoplasmic and stored Ca2+ in sensory neurons. Since influx of Ca2+ may occur through store-operated Ca2+ entry (SOCE) as well as voltage- and ligand-activated pathways, we sought confirmation of SOCE in sensory neurons from adult rats and examined whether dysfunction of SOCE is a possible pathogenic mechanism. Dorsal root ganglion neurons displayed a fall in resting cytoplasmic Ca2+ concentration when bath Ca2+ was withdrawn, and a subsequent elevation of cytoplasmic Ca2+ concentration (40 ± 5 nm) when Ca2+ was reintroduced, which was amplified by store depletion with thapsigargin (1 μm), and was significantly reduced by blockers of SOCE, but was unaffected by antagonists of voltage-gated membrane Ca2+ channels. We identified the underlying inwardly rectifying Ca2+-dependent ICRAC (Ca2+ release activated current), as well as a large thapsigargin-sensitive inward current activated by withdrawal of bath divalent cations, representing SOCE. Molecular components of SOCE, specifically STIM1 and Orai1, were confirmed in sensory neurons at both the transcript and protein levels. Axonal injury by spinal nerve ligation (SNL) elevated SOCE and ICRAC. However, SOCE was comparable in injured and control neurons when stores were maximally depleted by thapsigargin, and STIM1 and Orai1 levels were not altered by SNL, showing that upregulation of SOCE after SNL is driven by store depletion. Blockade of SOCE increased neuronal excitability in control and injured neurons, whereas injured neurons showed particular dependence on SOCE for maintaining levels of cytoplasmic and stored Ca2+, which indicates a compensatory role for SOCE after injury.

Painful Peripheral Nerve Injury Decreases Calcium Current in Axotomized Sensory Neurons

Background:Reports of Ca2+ current (ICa) loss after injury to peripheral sensory neurons do not discriminate between axotomized and spared neurons. The spinal nerve ligation model separates axotomized from spared neurons innervating the same site. The authors hypothesized that ICa loss is a result of neuronal injury, so they compared axotomized L5 dorsal root ganglion neurons to spared L4 neurons, as well as neurons from rats undergoing skin incision alone. Methods:After behavioral testing, dissociated neurons from L4 and L5 dorsal root ganglia were studied in both current and voltage patch clamp modes. The biophysical consequence of ICa loss on the action potential was confirmed using selective ICa antagonists. Data were grouped into small, medium, and large cells for comparison. Results:Reduced ICa was predominantly a consequence of axotomy (L5 after spinal nerve ligation) and was most evident in small and medium neurons. ICa losses were associated with action potential prolongation in small and medium cells, whereas the amplitude and duration of after hyperpolarization was reduced in medium and large neurons. Blockade with Ca2+ channel antagonists showed that action potential prolongation and after hyperpolarization diminution were alike, attributable to the loss of ICa. Conclusion:Axotomy is required for ICa loss. ICa loss correlated with changes in the biophysical properties of sensory neuron membranes during action potential generation, which were due to ICa loss leading to decreased outward Ca2+-sensitive K+ currents. Taken together, these results suggest that neuropathic pain may be mediated, in part, by loss of ICa and the cellular processes dependent on Ca2+.

Differential Modulation of the Cardiac Adenosine Triphosphate-sensitive Potassium Channel by Isoflurane and Halothane

Background The cardiac adenosine triphosphate–sensitive potassium (KATP) channel is activated during pathophysiological episodes such as ischemia and hypoxia and may lead to beneficial effects on cardiac function. Studies of volatile anesthetic interactions with the cardiac KATP channel have been limited. The goal of this study was to investigate the ability of volatile anesthetics halothane and isoflurane to modulate the cardiac sarcolemmal KATP channel. Methods The KATP channel current (IKATP) was monitored using the whole cell configuration of the patch clamp technique from single ventricular cardiac myocytes enzymatically isolated from guinea pig hearts. IKATP was elicited by extracellular application of the potassium channel openers 2,4-dinitrophenol or pinacidil. Results Volatile anesthetics modulated IKATP in an anesthetic-dependent manner. Isoflurane facilitated the opening of the KATP channel. Following initial activation of IKATP by 2,4-dinitrophenol, isoflurane at 0.5 and 1.3 mm further increased current amplitude by 40.4 ± 11.1% and 58.4 ± 20.6%, respectively. Similar results of isoflurane were obtained when pinacidil was used to activate IKATP. However, isoflurane alone was unable to elicit KATP channel opening. In contrast, halothane inhibited IKATP elicited by 2,4-dinitrophenol by 50.6 ± 5.8% and 72.1 ± 11.6% at 0.4 and 1.0 mm, respectively. When IKATP was activated by pinacidil, halothane had no significant effect on the current. Conclusions The cardiac sarcolemmal KATP channel is differentially modulated by volatile anesthetics. Isoflurane can facilitate the further opening of the KATP channel following initial channel activation by 2,4-dinitrophenol or pinacidil. The effect of halothane was dependent on the method of channel activation, inhibiting IKATP activated by 2,4-dinitrophenol but not by pinacidil.