Thomas S. McDowell

Local Anesthetic Inhibition of Voltage-activated Potassium Currents in Rat Dorsal Root Ganglion Neurons

Background Local anesthetic actions on the K+ channels of dorsal root ganglion (DRG) and dorsal horn neurons may modulate sensory blockade during neuraxial anesthesia. In dorsal horn neurons, local anesthetics are known to inhibit transient but not sustained K+ currents. The authors characterized the effects of local anesthetics on K+ currents of isolated DRG neurons. Methods The effects of lidocaine, bupivacaine, and tetracaine on K+ currents in isolated rat DRG neurons were measured with use of a whole cell patch clamp method. The currents measured were fast-inactivating transient current (IAf), slow-inactivating transient current (IAs), and noninactivating sustained current (IKn). Results One group of cells (type 1) expressed IAf and IKn. The other group (type 2) expressed IAs and IKn. The diameter of type 2 cells was smaller than that of type 1 cells. Lidocaine and bupivacaine inhibited all three K+ currents. Tetracaine inhibited IAs and IKn but not IAf. For bupivacaine, the concentration for half-maximal inhibition (IC50) of IKn in type 2 cells was lower than that for IKn in type 1 cells (57 vs. 121 &mgr;m). Similar results were obtained for tetracaine (0.6 vs. 1.9 mm) and for lidocaine (2.2 vs. 5.1 mm). Conclusions Local anesthetics inhibited both transient and sustained K+ currents in DRG neurons. Because K+ current inhibition is known to potentiate local anesthetic–induced impulse inhibition, the lower IC50 for IKn of small type 2 cells may reflect preferential inhibition of impulses in nociceptive neurons. The overall modulatory actions of local anesthetics probably are determined by their differential effects on presynaptic (DRG) and postsynaptic (dorsal horn neurons) K+ currents.

Functional properties of ryanodine receptors from rat dorsal root ganglia

The properties of ryanodine receptors (RyRs) from rat dorsal root ganglia (DRGs) have been studied. The density of RyRs (B max) determined by [3H]ryanodine binding was 63 fmol/mg protein with a dissociation constant (K d) of 1.5 nM. [3H]Ryanodine binding increased with caffeine, decreased with ruthenium red and tetracaine, and was insensitive to millimolar concentrations of Mg2+ or Ca2+. DRG RyRs reconstituted in planar lipid bilayers were Ca2+‐dependent and displayed the classical long‐lived subconductance state in response to ryanodine; however, unlike cardiac and skeletal RyRs, they lacked Ca2+‐dependent inactivation. Antibodies against RyR3, but not against RyR1 or RyR2, detected DRG RyRs. Thus, DRG RyRs are immunologically related to RyR3, but their lack of divalent cation inhibition is unique among RyR subtypes.

Volatile anesthetics reduce low-voltage-activated calcium currents in a thyroid C-cell line

Background Volatile anesthetics may act in part by inhibiting voltage-dependent calcium channels. The effects of several volatile agents on three types of calcium channels in a thyroid C-cell line were examined. Methods Whole-cell calcium currents were recorded using standard patch clamp techniques. Current-voltage relationships were derived before, during, and after application of isoflurane, enflurane, or halothane. Low-voltage-activated (LVA; T type) calcium currents were isolated based on the voltage range of activation. High-voltage-activated (HVA) calcium currents were separated into L and N types using omega-conotoxin GVIA (omega-CTX) and nicardipine. Results All three agents reversibly decreased both LVA and HVA currents at clinically relevant concentrations. Isoflurane and enflurane both reduced peak LVA current more than peak HVA current: -33 +/- 6% (mean +/- SE) versus -22 +/- 4% for 0.71 mM isoflurane (n = 6), and -46 +/- 6% versus -35 +/- 5% for 1.21 mM enflurane (n = 6). In contrast, halothane depressed LVA and HVA currents to a similar extent: -22 +/- 4% versus -29 +/- 3% for 0.65 mM halothane (n = 6). Isoflurane had no effect on LVA whole-cell current kinetics. Pretreatment with either omega-CTX (400 nM) or nicardipine (1 micro Meter) did not change the sensitivity of HVA current to isoflurane. Conclusions Isoflurane and enflurane block LVA calcium channels more potently than either L-type or N-type calcium channels, but halothane shows no such preferential effect. These results may have implications for the mechanism action of volatile anesthetics.

Intubation via the LMA using a Cook retrograde intubation kit

PurposeWe report two cases of difficult intubation where a laryngeal mask airway (LMA) was used and changed to a conventional endotracheal tube using a retrograde intubation set.Clinical findingsIn two patients, following induction of anaesthesia, the trachea could not be intubated in the conventional fashion with a blade. In both patients an LMA was inserted to achieve an airway. In both patients intubation with a conventional endotracheal tube was required. A Cook Retrograde Intubation Kit and fibreoptic bronchoscope were used to change the LMA to conventional endotracheal tube without problems.ConclusionThe Cook retrograde intubation allows an LMA to be replaced with an endotracheal tube with an ID greater than 6 mm with a #3 or 7 mm with a #5 LMA. This technique places an exchange stylet into the airway which is superior to a conventional guidewire. This method allows the airway to be maintained until the LMA is exchanged with an endotracheal tube. Anaesthesia may be maintained and the airway instrumented without difficulty using this technique.RésuméObjectifRapporter deux cas d’intubation difficile où un masque laryngé (ML) a d’abord été utilisé pour ensuite être remplacé par un tube endotrachéal conventionnel en utilisant une trousse d’intubation rétrograde.Observations cliniquesChez deux patients, suite à l’induction de l’anesthésie, l’intubation conventionnelle avec un laryngoscope s’est avérée impossible. Chez les deux patients, l’insertion d’un ML a permis d’obtenir une voie aérienne, mais une intubation conventionnelle était requise. Une trousse d’intubation rétrograde Cook et un fibroscope ont été utilisés pour remplacer sans difficulté le ML par un tube endotrachéal.ConclusionLa trousse d’intubation rétrograde Cook permet de remplacer un ML par un tube endotrachéal de diamètre interne plus grand que 6mm pour le ML #3 et plus grand que 7 mm pour le #5. Cette technique permet de placer dans les voies respiratoires un stylet d’échange supérieur à la broche guide conventionnelle. Cette méthode permet de maintenir les voies aériennes ouvertes jusqu’à ce que le ML puisse être remplacé par un tube endotrachéal; elle permet de poursuivre l’anesthésie et d’instrumenter sans difficulté les voies respiratoires.

Volatile anesthetic sensitivity of T-type calcium currents in various cell types.

UNLABELLED We evaluated the effects of volatile anesthetics on T-type calcium current (ICa,T) present in four different cell types using the whole cell version of the patch clamp technique. In dorsal root ganglion neurons and in two neuroendocrine cells--adrenal glomerulosa cells (AG) and thyroid C-cells--ICa,T was reversibly decreased by volatile anesthetics at clinically relevant concentrations, with isoflurane and enflurane being more potent that halothane. In AG cells, the most sensitive cell type tested, ICa,T was reduced 47%+/-4% (n = 6) by isoflurane (0.7 mM) and 56%+/-2% (n = 5) by enflurane (1.2 mM), but by only 24%+/-1% (n = 5; P < 0.05) by halothane (0.7 mM). Isoflurane caused a significant increase in the rate of deactivation of ICa,T in AG cells. In ventricular myocytes, however, ICa,T was much less sensitive to both isoflurane and halothane. The differential sensitivity of ICa,T in various cell types to the anesthetics may reflect differences in the channels expressed in these tissues or differences in the cellular intermediates involved in anesthetic action. Depression of ICa,T in neuronal cells may contribute to anesthetic action through decreases in cellular excitability. IMPLICATIONS Using the patch clamp technique, we showed that T-type calcium channels, which promote cellular excitability, are inhibited by volatile anesthetics in neuronal and neuroendocrine cells, but not in ventricular myocytes. Inhibition of neuronal T-type channels may contribute to the mechanism of action of volatile anesthetics.

Differential effects of bupivacaine and tetracaine on capsaicin-induced currents in dorsal root ganglion neurons

Capsaicin opens the TRPV1 channel, a cation channel that depolarizes and activates nociceptive neurons. Following this initial activation, neurons become desensitized to subsequent applications of capsaicin as well as to other noxious stimuli, a phenomenon attributed primarily to the entry of Ca2+ ions through the open TRPV1 channel. This ability of capsaicin to desensitize nociceptors has led to its use as an analgesic in the treatment of a variety of chronic pain states. Because treatment with capsaicin is initially quite painful, local anesthetics are sometimes used to block axonal conduction in nociceptive neurons and thus minimize pain. However, local anesthetics might also block TRPV1 and prevent the Ca2+ entry required for capsaicin-induced desensitization. We have studied the direct effect of local anesthetics on currents induced by capsaicin (1 microM) in acutely isolated rat dorsal root ganglion neurons using the whole cell patch clamp technique. At the highest concentration tested (1 mM), bupivacaine only moderately inhibited the capsaicin-induced current to 55 +/- 27% of control (mean +/- S.D.; n=12, p<0.01). Tetracaine (1 mM), on the other hand, enhanced the capsaicin-induced current to 151 +/- 34% of control (mean +/- S.D.; n=7, p<0.01). These results show that local anesthetics can be used to prevent the initial pain induced by application of capsaicin without abolishing, and perhaps even enhancing, its desensitizing actions.

Fentanyl decreases Ca2+ currents in a population of capsaicin-responsive sensory neurons.

Background Neuraxial opioids produce analgesia in part by decreasing excitatory neurotransmitter release from primary nociceptive neurons, an effect that may be due to inhibition of presynaptic voltage-activated Ca2+ channels. The purpose of this study was to determine whether opioids decrease Ca2+ currents (ICa) in primary nociceptive neurons, identified by their response to the algogenic agent capsaicin. Methods ICa was recorded from acutely isolated rat dorsal root ganglion neurons using the whole cell patch clamp technique before, during, and after application of the &mgr;-opioid agonist fentanyl (0.01–1 &mgr;m). Capsaicin was applied to each cell at the end of the experiment. Results Fentanyl reduced ICa in a greater proportion of capsaicin-responsive cells (62 of 106, 58%) than capsaicin-unresponsive cells (2 of 15, 13%;P < 0.05). Among capsaicin-responsive cells, the decrease in ICa was 38 ± 3% (n = 36, 1 &mgr;m) in fentanyl-sensitive cells versus just 7 ± 1% (n = 15, 1 &mgr;m;P < 0.05) in fentanyl-insensitive cells. Among capsaicin-responsive cells, ICa inactivated more rapidly in fentanyl-sensitive cells (&tgr;h, 52 ± 4 ms, n = 22) than in fentanyl-insensitive cells (93 ± 14 ms, n = 24;P < 0.05). This was not due to differences in the types of Ca2+ channels expressed as the magnitudes of &ohgr;-conotoxin GVIA-sensitive (N-type), nifedipine-sensitive (L-type), and GVIA/nifedipine-resistant (primarily P-/Q-type) components of ICa were similar. Conclusions The results show that opioid-sensitive Ca2+ channels are expressed by very few capsaicin-unresponsive neurons but by more than half of capsaicin-responsive neurons. The identity of the remaining capsaicin-responsive (and therefore presumed nociceptive) neurons that express opioid-insensitive Ca2+ channels is unknown but may represent a potential target of future non–opioid-based therapies for acute pain.

Exogenous nerve growth factor attenuates opioid-induced inhibition of voltage-activated Ba2+ currents in rat sensory neurons.

Nerve growth factor (NGF) promotes the survival of embryonic sensory neurons and maintains the phenotypic characteristics of primary nociceptive neurons postnatally. NGF also contributes to nociceptor activation and hyperalgesia during inflammatory pain states. The purpose of this study was to determine whether NGF might have an additional pronociceptive action by interfering with opioid-mediated analgesia in primary nociceptive neurons. Sensory neurons were isolated from the dorsal root ganglia of weanling rats and kept in standard culture conditions either with or without exogenous NGF (50 ng/ml). Currents through voltage-gated calcium channels were recorded from individual neurons using the whole cell patch clamp technique with Ba(2+) as the charge carrier (I(Ba)). The micro-opioid agonist fentanyl (1 microM) and the GABA(B) agonist baclofen (50 microM) were used to test G protein-dependent inhibition of I(Ba). Fentanyl inhibited I(Ba) by an average of 38+/-4% in untreated cells vs. 25+/-2% in NGF-treated cells (P<0.01). NGF had no effect on I(Ba) current magnitude or kinetics. The NGF-induced attenuation of opioid action was observed as early as 4 h after exposure, but was not seen when NGF was applied by bath perfusion for up to 40 min, suggesting that the effect was not mediated by a rapid phosphorylation event. The effect of NGF was prevented by K-252a (100 nM), an inhibitor of TrkA autophosphorylation. Baclofen-induced inhibition of I(Ba), on the other hand, was not affected by NGF treatment, suggesting that NGF modulation of opioid-mediated inhibition occurred upstream from the G protein. This was supported by the finding that GTP-gamma-S, an agonist independent G protein activator, inhibited I(Ba) similarly in both untreated and NGF treated cells. The results show that NGF selectively attenuated opioid-mediated inhibition of I(Ba) via TrkA receptor activation, possibly by altering opioid receptor function.

Effects of local anesthetics on opioid inhibition of calcium current in rat dorsal root ganglion neurons

Neuraxial analgesia is often provided using a mixture of local anesthetics and opioids. This combination of agents provides better pain relief and is generally associated with fewer side effects than when either drug is given alone. Local anesthetics have been shown to alter signaling of other G protein-coupled receptors, but little is known about their effect on opioid receptor signaling. Because opioids produce analgesia at least in part by inhibiting presynaptic Ca channels, we have evaluated the effects of tetracaine and bupivacaine on opioid-mediated inhibition of Ca channels in dorsal root ganglion neurons. The mu-opioid specific agonist DAMGO (1microM) inhibited Ca channels in both the absence and presence of tetracaine (50 or 100muM). However, the extent of DAMGO inhibition in the presence of both concentrations of tetracaine was less than that observed in the absence of tetracaine. DAMGO inhibition decreased from 39.2+/-24.4% in control to 34.2+/-24.4% with 50microM tetracaine (n=16; p<0.05), and from 40.5+/-19.6% in control to 34.6+/-20.5% with 100microM tetracaine (n=10; p<0.05). Similar results were seen with bupivacaine. Tetracaine also decreased the voltage-dependent facilitation of Ca channel currents when G proteins were activated by either DAMGO or the non-hydrolyzable GTP analogue (GTPgammaS), suggesting that tetracaine weakens the interaction between G protein betagamma subunits and the Ca channel. Overall, these results suggest that local anesthetics decrease opioid inhibition of Ca channel activity by interfering with the GTP-mediated signal transduction between opioid receptors and Ca channels.

HSP90 inhibitors alter capsaicin- and ATP-induced currents in rat dorsal root ganglion neurons.

Heat shock proteins (HSPs) are major components of eukaryotic and prokaryotic cells with particularly high levels of expression in neurons. HSPs control protein folding, transport of proteins to and from the nucleus, incorporation of proteins into the cell membrane, and maintenance of the functional activity of several proteins involved in transcriptional control. In this study we demonstrate that inhibitors of HSP90 alter currents mediated by the ligand gated channels, P2X and VR1. P2X and VR1 are membrane receptors activated by ATP and capsaicin, respectively, and are thought to be involved in inflammation-related nociception. The HSP90 inhibitors geldanamycin (GLD), radicicol (RAD) herbimycin A (HERB) potentiated ATP induced currents, whereas only GLD altered capsaicin-induced currents in isolated DRG neurons. At low (< 1μM) concentrations, GLD potentiated the capsaicin-induced current, while at high concentrations (10–25 μM) it inhibited it. The results suggest a potential involvement of HSPs in nociception.