The effects of isoflurane at 0.3 mM (approximately 1 MAC) with those of F8 (8.8 μM approximately 1 MACeq) and F6 (35.6 μM approximately 2 MACeq) on CaV1 single-channel currents in SCMN were compared. Figure 3 shows single-channel current traces at various holding potentials and the corresponding single-channel current-voltage relationship that gave a single-channel conductance of approximately 21pS, a property consistent with CaV1 single-channel currents. Figure 3B demonstrates single-channel currents before, during, and after exposure to 0.3 mM, approximately 1 MAC, isoflurane. CaV1 currents were statistically significantly inhibited by 0.3 mM (approximately 1 MAC) isoflurane, P = 0.0002 (Fig. 4), whereas F6, P = 0.9240, and F8, P = 0.3958, at 1 and 2 MACeq, respectively, had no significant effects (Fig. 4).
In DRGN Nonimmobilizers Inhibit CaV2 (N-Type) and CaV1 (L-Type) Calcium Currents
CaV2 current traces in DRGN were measured at +10 mV, and their corresponding current-voltage relationships for a control cell (Fig. 5A) and for cells before, during drug exposure (isoflurane or F6), and after drug removal (Fig. 5, B and C) are shown. F6, P < 0.0001, significantly inhibited CaV2 currents in DRGN (Fig. 6), whereas the effect of isoflurane was not consistent, P = 0.037.
In DRGN, we found that isoflurane at approximately 2 MAC (0.6 mM), P = 0.0081, and F6 at approximately 2 MACeq (35.6 μM), P = 0.0117, but not F8 (17.6 μM), P = 0.3326, significantly inhibited CaV1 currents (Fig. 7). Although the sample size was small, Lilliefors and Shapiro Wilk tests of normality and the Levene test for equal variance of the residuals of the analysis of variance for drug administration and drug administration plus the wash period, respectively, demonstrated normal distributions and equal variances as indicated in the legend of Figure 7. Because F6 had an inhibitory effect on CaV2 and CaV1 currents in DRGN, these channels in DRGN are unlikely to be involved in anesthetic-induced immobility.
SCMN and DRGN were exposed to the nonimmobilizers, F6 and F8, to determine whether CaV1 and/or CaV2 channels in these neurons might be involved in isoflurane-induced immobility. CaV1 channels in SCMN were strongly inhibited at a clinically relevant isoflurane concentration (80% inhibition at 0.3 mM isoflurane approximately 1 MAC) but were not inhibited by F6 or F8 at their 1 or 2 MACeq, suggesting that SCMN CaV1 channels might contribute to isoflurane-induced immobility. However, SCMN CaV2 channels were strongly inhibited by F6 and F8 but not by isoflurane (at 2 MAC), suggesting that CaV2 channels do not contribute to isoflurane-induced immobility. Furthermore, CaV1 and CaV2 Ca2+ channels in rat DRGN were inhibited by F6, suggesting that CaV1 and CaV2 channels in DRGN do not contribute to isoflurane-induced immobility.
It is well known that CaV2 and CaV1 calcium channel functions are highly regulated by post-translational modifications (e.g., phosphorylation), as well as by interactions with other cellular proteins, for example, G-proteins; hence, the different responses of a given voltage-dependent calcium channel in SCMN and DRGN could reflect differences not only in their structure but also in modulation by other cellular processes.5,27 Regardless of the possible underlying structural or regulatory mechanisms that lead to the different responses to isoflurane and nonimmobilizers for a given channel type in different neuronal cells, our results suggest that calcium signaling, and specifically CaV1 calcium channels in SCMN, may be involved in the mechanism of isoflurane-induced immobility.
AMPA (a-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid), NMDA (N-methyl-D-aspartate), glycine, and GABAA receptors are reportedly involved in the overall anesthetic effect,28,29 but other receptors and channels may also be relevant. It has been shown that enflurane at 1 MAC depressed synaptic currents in SCMN.30 A paradigm involving multiple effects of VAs on several ligands and channels has been described that could explain how different ligand and receptors, including voltage-dependent calcium channels, may participate in isoflurane-induced immobility.31 Our data support CaV1 channels in SCMN as one of several targets involved in VA-induced immobility. It is not yet clear how CaV1 channels are modulated by VAs, which can act directly on the CaV1 channel or indirectly through secondary effectors. It is known that isoflurane can act on the channel by increasing current inactivation and prolonging recovery time after inactivation; for example, halothane inhibits CaV1 calcium channels in SH-SY5Y cells through stabilization of nonconducting or inactivated states.4 We also have shown that activation of G-proteins reduces isoflurane inhibition of CaV2 calcium currents in SH-SY5Y cells5 and in DRGN (unpublished data).
Inhibition of CaV1 calcium channels by VAs has been demonstrated. Recombinant cardiac CaV1 calcium channels are inhibited by halothane.2 CaV1 calcium channels are involved in neurotransmission, gene expression, and regulating neuronal excitability.32,33 CaV1 calcium currents mediate rhythmic activity induced by a cholinergic antagonist in motor neurons, suggesting that the entry of calcium through CaV1 calcium channels is a significant pathway involved in agonist-modulated locomotor activity.34 CaV1 calcium channels also contribute to NMDA-induced intrinsic oscillations in mature turtle SCMNs.35 There are also studies showing the effects of VAs on calcium regulation and specifically on voltage-dependent calcium channels.36 Our results direct attention to CaV1 channels in SCMN as a relevant target of VA-induced immobility. The action of the nonimmobilizer F6 on rat DRGN, but not rat SCMN CaV1 calcium channels, suggests that these channels in DRGN are not a prominent site of isoflurane-induced immobility.15,19 Furthermore, the inhibition by isoflurane of CaV1 channels in rat SCMN, but not the nonimmobilizers, supports the work of others describing anesthetic-induced immobility as an effect mediated through the ventral spinal cord.17–19
Using the lamprey spinal cord preparation, Jinks et al.37 concluded that ventral SCMN involved in central pattern locomotor activity were an important site of isoflurane-induced immobility. They subsequently used electrical microstimulation of the mesencephalic locomotor region in decerebrated rats and determined that VAs produce “immobility mainly by action on ventral spinal cord locomotor networks.”15 However, motor neurons themselves have been shown to express persistent oscillatory activity sensitive to CaV1 and CaV2 Ca2+ channels and are potentially related to locomotor networks.34,35,38–41 SCMN express CaV1.2,3(L-), CaV2.1(N-), CaV2.2(P/Q), CaV2.3(R), and CaV3(T-)type channels, but mainly CaV1.2,.3(L-) and CaV2.1(N-)type.42 Both CaV1.2 and CaV1.3 L-type channels are found in the CNS, and CaV1.3 (α1D) channels are involved in persistent inward currents, which give SCMN the property of bistability and sustained activation.39,41,43
On the basis of electromyographic studies in the rat, King and Rampil17 suggested that isoflurane can depress SCMN. They stimulated the tibial nerve of rats and measured the amplitude of the orthodromic (M-wave) and antidromic (F-wave) electromyogram and showed that only the F-wave was inhibited by isoflurane.17,18 The F-wave results from the antidromic transmission back to the motor neuron followed by orthodromic transmission from the motor neuron. The CaV1 calcium channel is found predominantly in the soma but also in the proximal dendrites of rat SCMN.44,45 The action of isoflurane to inhibit calcium movement through the CaV1 channel could result in a higher threshold for activating an action potential, resulting in inhibition of neurotransmission. The CaV1 channel in the motor neuron has approximately a 75% homogeneity with the CaV1 channel in skeletal muscle; yet, as shown in the studies of Rampil, although the F-wave is markedly depressed by isoflurane, the M-wave, representing direct stimulation of the motor endplate, was unaffected by isoflurane. That result suggests that the CaV1 channel of the motor neuron and skeletal muscle is sufficiently different that, at the clinically relevant concentrations studied, isoflurane had little effect on the skeletal muscle CaV1 calcium channel, despite inhibiting calcium movement through CaV1 channels in the SCMN.
Although we have presented evidence that CaV1 channels in SCMN are a likely target for isoflurane-induced immobility, several limitations must be considered. Our studies were performed in vitro on cultured DRGN and SCMN. Although the identified channels demonstrated characteristics of CaV1 and CaV2 channels, we cannot rule out the possibility that the isolation process might have altered the channels’ response to isoflurane and nonimmobilizers. A further limitation is that the concentrations of isoflurane and F6 and F8 in the solutions bathing the SCMN and DRGN were not measured. However, recordings were performed while the chamber was continuously perfused; moreover, the observed inhibitory effects of F6 and F8, on CaV2 channels in SCMN, and CaV1 and CaV2 channels in DRGN support our methodology and suggest that F6 and F8 were present in the chamber solutions used to examine CaV1 channels, despite there being no effect on these channels. Finally, we studied only one VA, isoflurane. Is it unique in its target or do other VAs also induce immobility by targeting the SCMN CaV1 channels? There is supporting evidence in other cell types, including primary cultures and expression systems, that some of these VAs can alter CaV1 function; however, none of them include CaV1 channels from SCMN. Whether CaV1 in SCMN are also targeted by other VAs, such as halothane, enflurane, desflurane and sevoflurane, remains to be determined.
The physiologic implication of our findings is related to the fact that calcium contributes to the regulation of neuronal excitability through its actions on many sites within a cell.46,47 Activation of CaV1 calcium channels leads to increased excitability of neurons, whereas inhibition of CaV1 channels can lead to decreased excitability. VAs, including isoflurane, have multiple effects on Ca2+ homeostasis in neuronal cells. The proposed isoflurane-induced decreased excitability of motor neurons can be related not only to CaV1 channel inhibition but might also involve modification of Na+ channel conductance and other ion channel conductances involved in intracellular Ca2+ regulation. In summary, we found that CaV1 calcium channels in SCMN could contribute to isoflurane-mediated immobility. Additional studies are required to examine whether inhibition of CaV1 calcium channels is sufficient or whether isoflurane actions on other channels in SCMN also contribute to isoflurane-induced immobility.
Name: Esperanza Recio-Pinto, PhD.
Contribution: This author helped design the study, conduct the study, analyze the data, and write the manuscript.
Attestation: Esperanza Recio-Pinto has seen the original study data, reviewed the analysis of the data, approved the final manuscript, and is the author responsible for archiving the study files.
Name: Jose V. Montoya-Gacharna, MD.
Contribution: This author helped design the study, conduct the study, analyze the data, and write the manuscript.
Attestation: Jose V. Montoya-Gacharna has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.
Name: Fang Xu, PhD.
Contribution: This author helped design the study, write the manuscript, and served as a technical advisor for the experiments.
Attestation: Fang Xu has seen the original study data and approved the final manuscript
Name: Thomas J. J. Blanck, MD, PhD.
Contribution: This author helped design the study, analyze the data, and write the manuscript.
Attestation: Thomas J. J. Blanck has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.
This manuscript was handled by: Markus W. Hollmann, MD, PhD, DEAA.
1. Zhang J, Sutachan JJ, Montoya-Gacharna J, Xu CF, Xu F, Neubert TA, Recio-Pinto E, Blanck TJ. Isoflurane inhibits cyclic adenosine monophosphate response element-binding protein phosphorylation and calmodulin translocation to the nucleus of SH-SY5Y cells. Anesth Analg. 2009;109:1127–34
2. Gingrich KJ, Tran S, Nikonorov IM, Blanck TJ. Halothane inhibition of recombinant cardiac L-type Ca2+ channels expressed in HEK-293 cells. Anesthesiology. 2005;103:1156–66
3. Herrington J, Lingle CJ. Halothane reduces calcium currents in clonal (GH3) pituitary cells. Ann N Y Acad Sci. 1991;625:290–2
4. Nikonorov IM, Blanck TJ, Recio-Pinto E. The effects of halothane on single human neuronal L-type calcium channels. Anesth Analg. 1998;86:885–95
5. Nikonorov IM, Blanck TJ, Recio-Pinto E. G-protein activation decreases isoflurane inhibition of N-type Ba2+ currents. Anesthesiology. 2003;99:392–9
6. Study RE. Isoflurane inhibits multiple voltage-gated calcium currents in hippocampal pyramidal neurons. Anesthesiology. 1994;81:104–16
7. Eilers H, Kindler CH, Bickler PE. Different effects of volatile anesthetics and polyhalogenated alkanes on depolarization-evoked glutamate release in rat cortical brain slices. Anesth Analg. 1999;88:1168–74
8. Kress HG, Müller J, Eisert A, Gilge U, Tas PW, Koschel K. Effects of volatile anesthetics on cytoplasmic Ca2+ signaling and transmitter release in a neural cell line. Anesthesiology. 1991;74:309–19
9. Schlame M, Hemmings HC Jr. Inhibition by volatile anesthetics of endogenous glutamate release from synaptosomes by a presynaptic mechanism. Anesthesiology. 1995;82:1406–16
10. Wu XS, Sun JY, Evers AS, Crowder M, Wu LG. Isoflurane inhibits transmitter release and the presynaptic action potential. Anesthesiology. 2004;100:663–70
11. Dodge FA Jr, Rahamimoff R. Co-operative action a calcium ions in transmitter release at the neuromuscular junction. J Physiol. 1967;193:419–32
12. Augustine GJ, Charlton MP, Smith SJ. Calcium entry and transmitter release at voltage-clamped nerve terminals of squid. J Physiol. 1985;367:163–81
13. Antognini JF. The relationship among brain, spinal cord and anesthetic requirements. Med Hypotheses. 1997;48:83–7
14. Antognini JF, Schwartz K. Exaggerated anesthetic requirements in the preferentially anesthetized brain. Anesthesiology. 1993;79:1244–9
15. Jinks SL, Bravo M, Hayes SG. Volatile anesthetic effects on midbrain-elicited locomotion suggest that the locomotor network in the ventral spinal cord is the primary site for immobility. Anesthesiology. 2008;108:1016–24
16. Rampil IJ, Mason P, Singh H. Anesthetic potency (MAC) is independent of forebrain structures in the rat. Anesthesiology. 1993;78:707–12
17. King BS, Rampil IJ. Anesthetic depression of spinal motor neurons may contribute to lack of movement in response to noxious stimuli. Anesthesiology. 1994;81:1484–92
18. Rampil IJ, King BS. Volatile anesthetics depress spinal motor neurons. Anesthesiology. 1996;85:129–34
19. Kim J, Yao A, Atherley R, Carstens E, Jinks SL, Antognini JF. Neurons in the ventral spinal cord are more depressed by isoflurane, halothane, and propofol than are neurons in the dorsal spinal cord. Anesth Analg. 2007;105:1020–6
20. Rampil IJ. Anesthetic potency is not altered after hypothermic spinal cord transection in rats. Anesthesiology. 1994;80:606–10
21. Koblin DD, Chortkoff BS, Laster MJ, Eger EI II, Halsey MJ, Ionescu P. Polyhalogenated and perfluorinated compounds that disobey the Meyer-Overton hypothesis. Anesth Analg. 1994;79:1043–8
22. Catterall WA. Voltage-gated calcium channels. Cold Spring Harb Perspect Biol. 2011;3:a003947
23. Castillo C, Norcini M, Baquero-Buitrago J, Levacic D, Medina R, Montoya-Gacharna JV, Blanck TJ, Dubois M, Recio-Pinto E. The N-methyl-D-aspartate-evoked cytoplasmic calcium increase in adult rat dorsal root ganglion neuronal somata was potentiated by substance P pretreatment in a protein kinase C-dependent manner. Neuroscience. 2011;177:308–20
24. Montoya-Gacharna JV, Sutachan JJ, Chan WS, Sideris A, Blanck TJ, Recio-Pinto E. Preparation of adult spinal cord motor neuron cultures under serum-free conditions. Methods Mol Biol. 2012;846:103–16
25. Xu F, Zhang J, Recio-Pinto E, Blanck TJ. Halothane and isoflurane augment depolarization-induced cytosolic Ca2+ transients and attenuate carbachol-stimulated Ca2+ transients. Anesthesiology. 2000;92:1746–56
26. Cardoso RA, Yamakura T, Brozowski SJ, Chavez-Noriega LE, Harris RA. Human neuronal nicotinic acetylcholine receptors expressed in Xenopus oocytes predict efficacy of halogenated compounds that disobey the Meyer-Overton rule. Anesthesiology. 1999;91:1370–7
27. Calin-Jageman I, Lee A. Ca(v)1 L-type Ca2+ channel signaling complexes in neurons. J Neurochem. 2008;105:573–83
28. Sonner JM, Zhang Y, Stabernack C, Abaigar W, Xing Y, Laster MJ. GABA(A) receptor blockade antagonizes the immobilizing action of propofol but not ketamine or isoflurane in a dose-related manner. Anesth Analg. 2003;96:706–12
29. Duarte LT, Saraiva RA. [Immobility: essential inhalational anesthetics action.]. Rev Bras Anestesiol. 2005;55:100–17
30. Cheng G, Kendig JJ. Enflurane directly depresses glutamate AMPA and NMDA currents in mouse spinal cord motor neurons independent of actions on GABAA or glycine receptors. Anesthesiology. 2000;93:1075–84
31. Eger EI II, Raines DE, Shafer SL, Hemmings HC Jr, Sonner JM. Is a new paradigm needed to explain how inhaled anesthetics produce immobility? Anesth Analg. 2008;107:832–48
32. Spedding M, Kenny B, Chatelain P. New drug binding sites in Ca2+ channels. Trends Pharmacol Sci. 1995;16:139–42
33. Rubi L, Schandl U, Lagler M, Geier P, Spies D, Gupta KD, Boehm S, Kubista H. Raised activity of L-type calcium channels renders neurons prone to form paroxysmal depolarization shifts. Neuromolecular Med. 2013;15:476–92
34. Guertin PA, Hounsgaard J. L-type calcium channels but not N-methyl-D-aspartate receptor channels mediate rhythmic activity induced by cholinergic agonist in motoneurons from turtle spinal cord slices. Neurosci Lett. 1999;261:81–4
35. Guertin PA, Hounsgaard J. NMDA-induced intrinsic voltage oscillations depend on L-type calcium channels in spinal motoneurons of adult turtles. J Neurophysiol. 1998;80:3380–2
36. Bleakman D, Jones MV, Harrison NL. The effects of four general anesthetics on intracellular [Ca2+] in cultured rat hippocampal neurons. Neuropharmacology. 1995;34:541–51
37. Jinks SL, Atherley RJ, Dominguez CL, Sigvardt KA, Antognini JF. Isoflurane disrupts central pattern generator activity and coordination in the lamprey isolated spinal cord. Anesthesiology. 2005;103:567–75
38. Büschges A, Wikström MA, Grillner S, El Manira A. Roles of high-voltage-activated calcium channel subtypes in a vertebrate spinal locomotor network. J Neurophysiol. 2000;84:2758–66
39. Carlin KP, Jones KE, Jiang Z, Jordan LM, Brownstone RM. Dendritic L-type calcium currents in mouse spinal motoneurons: implications for bistability. Eur J Neurosci. 2000;12:1635–46
40. Hsiao CF, Del Negro CA, Trueblood PR, Chandler SH. Ionic basis for serotonin-induced bistable membrane properties in guinea pig trigeminal motoneurons. J Neurophysiol. 1998;79:2847–56
41. Zhang M, Sukiasyan N, Møller M, Bezprozvanny I, Zhang H, Wienecke J, Hultborn H. Localization of L-type calcium channel Ca(V)1.3 in cat lumbar spinal cord–with emphasis on motoneurons. Neurosci Lett. 2006;407:42–7
42. Carlin KP, Jiang Z, Brownstone RM. Characterization of calcium currents in functionally mature mouse spinal motoneurons. Eur J Neurosci. 2000;12:1624–34
43. Heckmann CJ, Gorassini MA, Bennett DJ. Persistent inward currents in motoneuron dendrites: implications for motor output. Muscle Nerve. 2005;31:135–56
44. Hell JW, Westenbroek RE, Warner C, Ahlijanian MK, Prystay W, Gilbert MM, Snutch TP, Catterall WA. Identification and differential subcellular localization of the neuronal class C and class D L-type calcium channel alpha 1 subunits. J Cell Biol. 1993;123:949–62
45. Westenbroek RE, Hoskins L, Catterall WA. Localization of Ca2+ channel subtypes on rat spinal motor neurons, interneurons, and nerve terminals. J Neurosci. 1998;18:6319–30
46. Takahashi T, Momiyama A. Different types of calcium channels mediate central synaptic transmission. Nature. 1993;366:156–8
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47. Hille B Ionic Channels of Excitable Membranes. 19922nd ed Sunderland, MA Sinauer Associates Inc.