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Anesth Analg 2003;97:1020-1024
© 2003 International Anesthesia Research Society

ANESTHETIC PHARMACOLOGY

The Use of the Potassium Channel Activator Riluzole to Test Whether Potassium Channels Mediate the Capacity of Isoflurane to Produce Immobility

Yilei Xing, MD, Yi Zhang, MD, Caroline R. Stabernack, MD, Edmond I Eger, II, MD, and Andrew T. Gray, MD PhD

Department of Anesthesia and Perioperative Care, University of California, San Francisco, California

Address correspondence to Andrew T. Gray, MD, PhD, Department of Anesthesia and Perioperative Care, Room 3C-38, San Francisco General Hospital, University of California, San Francisco, CA 94110. Address e-mail to graya{at}anesthesia.ucsf.edu


   Abstract
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 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
Inhaled anesthetics produce immobility during noxious stimulation, primarily by actions on the spinal cord. In this study, we examined whether activation of potassium channels of the KCNK subfamily alters volatile anesthetic potency. We measured the change in isoflurane minimum alveolar anesthetic concentration (MAC) during 4-h intrathecal or IV infusions of the nonspecific KCNK activator riluzole in 54 Sprague-Dawley rats. IV or intrathecal infusions of riluzole doses that did not result in permanent injury or death equally decreased isoflurane MAC. We conclude that although riluzole exhibited anesthetic effects, the similar dose response from IV or intrathecal infusion suggests systemic absorption and actions in the brain rather than the spinal cord.

IMPLICATIONS: Riluzole, a drug that activates potassium channels and decreases glutamatergic neurotransmission, primarily acts on supraspinal sites to produce immobility in response to noxious stimuli. This finding does not support the hypothesis that potassium channels mediate the capacity of inhaled anesthetics to produce immobility in the face of noxious stimulation.


   Introduction
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 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
The human genome encodes >60 potassium channel subunits. Of these subunits, 14 contain 4 transmembrane domains flanking 2 pore loops in tandem within their primary amino acid sequence (1). This subfamily of potassium channel subunits (known formally as the KCNK subfamily) has been suggested as a potential target of inhaled anesthetic action (2). Consistent with this hypothesis, application of volatile anesthetics enhances the currents carried by ion channels formed by several of these subunits. These subunits are TREK-1 (KCNK2), TREK-2 (KCNK10), TASK-1 (KCNK3), TASK-2 (KCNK5), and TASK-3 (KCNK9). Because potassium channels modulate membrane potential, activation by anesthetics could suppress neuronal excitability. Supporting this concept, halothane hyperpolarizes motoneurons in spinal cord and brainstem preparations (3–6).

Riluzole is a benzothiazole used to treat patients with amyotrophic lateral sclerosis (7). It potently (20–100 µM) activates the mechano-gated KCNK channels TREK-1, TREK-2, and TRAAK (8,9) and less potently inhibits voltage-gated sodium channels. Riluzole can depress glutamatergic neurotransmission, is neuroprotective in several models of ischemia (10–13), and protects spinal motoneurons from ischemic insults (14). Riluzole decreases presynaptic glutamate release, and intraperitoneal injection can decrease the halothane minimum alveolar concentration preventing movement in response to a noxious stimulus in 50% of animals (MAC) (15,16).

Several research groups have proposed KCNK channels as relevant targets of volatile anesthetic action (17–20). Volatile anesthetics activate KCNK channels expressed in important neuroanatomic sites, including spinal cord. Effects on the spinal cord are particularly pertinent because the spinal cord is thought to mediate the capacity of inhaled anesthetics to produce immobility (21–23). KCNK channels closely resemble potassium channels known to reversibly hyperpolarize and inhibit mammalian neurons. Thus, activation of baseline potassium channels of the KCNK family appears to be a plausible mechanism of inhaled anesthetic action. However, other investigators have found but small effects of volatile anesthetics—effects that occur only at supratherapeutic levels (24). These results show the dependence of the heterologous expression system and question the extent of volatile anesthetic activation of KCNK channels expressed by native cells.

In this study, we took a pharmacological approach to determine what role, if any, that KCNK subunits have in the capacity of volatile anesthetics to produce immobility. Using intrathecal and IV infusions of riluzole, we tested the hypothesis that KCNK subunits contribute to MAC. We hypothesized that if KCNK channels mediate immobility, then infusion of riluzole should decrease MAC and that intrathecal infusion should decrease MAC more potently than IV infusion.


   Methods
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 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Our methods followed those described by Zhang et al. (25,26). With approval of the Committee on Animal Research of the University of California, San Francisco, we studied 2- to 3-mo-old male specific-pathogen-free Sprague-Dawley rats [Crl:CD®(SD)Br] obtained from Charles River Laboratories (Hollister, CA). Each animal was used in only one experiment. Intrathecal or IV catheters were inserted under isoflurane anesthesia by methods described by Yaksh and Rudy (27) and Zhang et al. (25,26). Rats were allowed to recover from anesthesia and surgery for at least 24 h before study.

The isoflurane MAC was determined in groups of 4 to 10 rats before and after infusion of drug or vehicle control (which included dimethyl sulfoxide (DMSO)). Each rat was placed in a clear plastic cylinder. A rectal temperature probe was inserted to allow control of body temperature (37°C–38°C) by using a heat lamp or application of ice to the plastic chambers as needed.

During determination of the initial (control) MAC (MAC0), we infused artificial cerebrospinal fluid (aCSF) through the intrathecal catheter at 1 µL/min or saline through the IV catheter at 10 µL/min. Isoflurane was introduced into the system at a partial pressure of approximately 1.0% of an atmosphere. Isoflurane partial pressures were monitored with an infrared analyzer (Datascope, Helsinki, Finland), but the concentration used in determinations of MAC was obtained with gas chromatography. After a 30-min equilibration period, a tail clamp was applied to the proximal portion of the tail and oscillated 45° at approximately 1 Hz for 1 min or until the animal moved (whichever came first). All animals moved at the initial isoflurane partial pressure. The isoflurane partial pressure was then increased in 0.1% to 0.2% atmosphere steps (30 min per step) until the isoflurane partial pressures bracketing movement and lack of movement during application of the tail-clamp stimulus were determined.

To determine MAC1, we then infused study drug or vehicle control at 1–4 µL/min through the intrathecal catheter or at 10–40 µL/min through the IV catheter. These rates were chosen because postmortem examination in previous studies indicated that infusions of methylene blue at such rates did not spread beyond the lumbar and lower thoracic spinal cord (28). Again allowing 30 min for each equilibration step, the isoflurane partial pressure was decreased by 0.1% to 0.2% atmospheres until the isoflurane partial pressures bracketing lack of movement and movement during the tail-clamp stimulus were determined.

To determine MAC2, we next increased isoflurane partial pressure in 0.1% to 0.2% atmosphere steps (30 min per step) until the isoflurane partial pressures bracketing movement and lack of movement during application of the tail-clamp stimulus were determined. Infusions continued throughout the determinations of MAC1 and MAC2 (i.e., for at least 4 h.) The calculated change in MAC2 was compared with that obtained for MAC1. This allowed an estimate of whether a steady-state drug effect was achieved with infusion.

Finally, in several studies we allowed the rats to recover from anesthesia for at least 24 h. We then redetermined MAC (recovery MAC; MACr). A comparison of MACr with MAC0 allowed an assessment of whether damage to the spinal cord had occurred.

MAC values were determined by a single crossover: the mean of the least partial pressure that caused immobility and the greatest partial pressure that allowed movement. Change in MAC was calculated as the percent change relative to the initial value, MAC0. That is, for MAC2, the percent change in MAC equaled 100 x (MAC2 - MAC0)/MAC0.

For IV infusions, movement of the head, forelimbs, or hindlimbs was considered a positive response. For intrathecal infusions, movement of the head or forelimbs or movement of the hindlimbs was considered separately as a positive response.

The aCSF was made daily from stock solutions. The final composition of the aCSF was 154.7 mM Na+, 0.82 mM Mg2+, 2.9 mM K+, 132.49 mM Cl-, 1.1 mM Ca2+, and 5.9 mM glucose (pH 7.4). We used the fastest intrathecal infusion rates (1–4 µL/min) that confine drug delivery to the lumbothoracic cord of rats. This rate was chosen because postmortem examination in previous studies using this infusion rate and duration of study established that aCSF containing methylene blue did not spread beyond the lumbothoracic cord.

Riluzole (RP 54274; 2-amino-6-trifluoromethoxybenzothiazole; Sigma, St. Louis, MO) solid powder was stored at room temperature and was dissolved in DMSO at 25 mg/mL (107 mM). This dissolved riluzole was diluted in saline to produce various concentrations of riluzole in DMSO. DMSO concentrations ranged from 10% to 25%. We did not exceed 25% because preliminary studies demonstrated that larger concentrations of DMSO (e.g., 37.5%–75%) combined with riluzole produced cord injury.

Differences in volatile anesthetic potency between control and experimental groups were compared with two-sample unpaired or paired Student’s t-tests. P < 0.05 was considered statistically significant. Animals that were obviously ill (n = 1) or with necropsy-proven intramedullary catheters (n = 4) were excluded from analysis. One animal died before MACr experiments could be performed. Necropsy did not reveal an obvious cause of death.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Isoflurane MAC0 values were significantly (P < 0.01) smaller in animals with intrathecal catheters (1.35% ± 0.13%; n = 16) than those with IV catheters (1.48% ± 0.14%; n = 38). IV and intrathecal control infusions of DMSO (the same vehicle as used for the corresponding infusion of riluzole) did not affect MAC (Table 1). Intrathecal and IV infusions of riluzole up to 9 µg/min (Table 1) minimally influenced MAC (Table 1, Fig. 1). Larger infusions decreased MAC. At the largest IV infusion (50 µg/min), all animals ultimately died of respiratory depression, and no MACr was obtained.


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  		Table 1. Effect of IV Versus Intrathecal (IT) Administration of Riluzole
 


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  		Figure 1. Intrathecal ({blacklozenge}) and IV ({circ}) infusions of riluzole change the isoflurane minimum alveolar anesthetic concentration (MAC) by similar amounts. At infusion rates of 9 µg/min, neither intrathecal nor IV riluzole administration materially affects MAC. At 12.5 µg/min, both significantly decrease MAC by the same amount. Increased rates of intrathecal infusion produce cord injury. The fastest rate of IV infusion (50 µg/min) ultimately produces respiratory depression and death. Note that the box and rectangle in the graph separate the control (no riluzole) from the riluzole data. This separation is required by the use of a logarithmic scale for the abscissa.

 
Isoflurane MAC1 and MAC2 values did not differ for riluzole infusions of <=12.5 µg/min. For rats given riluzole 25 or 50 µg/min IV, the decrease in MAC2 exceeded that for MAC1 (P < 0.05; paired Student’s t-test). Isoflurane MACr did not differ from initial measurements (MAC0; Table 1). Finally, MAC as determined by movement of the hindlimbs did not differ from MAC as determined by movement of the forelimbs (Fig. 2).



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  		Figure 2. As shown by this linear regression, intrathecal riluzole administration equally affects movement by hindlimbs and forelimbs. Data shown include vehicle control data, data for intrathecal infusion rates of 25 µg/min (which resulted in permanent injury), and data for intermediate infusion rates.

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
Consistent with our hypothesis, we found that riluzole decreased the MAC of isoflurane. This confirms the report that intraperitoneal injection can decrease halothane MAC (15,16). However, contradicting our hypothesis, we found no difference in the decrease in MAC produced by intrathecal versus IV riluzole (Table 1, Fig. 1). These results indicate that riluzole does not have a spinal site of anesthetic action. Because intrathecal infusion of riluzole was no more effective than IV infusion, the effect of intrathecal infusion likely resulted from systemic absorption and actions on higher centers. Consistent with this notion, and with modulation of sensory or supraspinal pathways, the estimates of change in isoflurane MAC values for hindlimbs and forelimbs did not differ (Fig. 2).

The anesthetic action of riluzole may result from inhibition of synaptic glutamate release (9). In addition, riluzole may block glutamatergic neurotransmission by direct blockade of ionotropic glutamate receptors, inactivation of voltage-dependent sodium and calcium channels, and activation of KCNK channels. These mechanisms may act synergistically at glutamatergic synapses. Glutamate receptor antagonists may alter volatile anesthetic potency through actions on the spinal cord (29), but this study suggests that the anesthetic action of riluzole is via inhibition of glutamatergic neurotransmission at supraspinal sites (e.g., as reported for the hippocampus) (30).

Rats recovered uneventfully from smaller IV doses of riluzole, but large IV doses of riluzole (50 µg/min) produced respiratory depression and death. Intrathecal infusion of riluzole at doses larger than 12.5 µg/min required DMSO concentrations in excess of 25% to dissolve the riluzole. This combination caused injury to the cord, as evidenced by loss of motor function in the hindlimbs, and these data are not included in this report. DMSO vehicle controls (DMSO alone) altered MAC at the largest concentration (75%) tested (again, data not included).

Riluzole, at 5 mg/kg intraperitoneally, decreases halothane MAC by 40% (15). In this study, with a 12.5 µg/min IV infusion (approximately 10 mg/kg over the study period), we observed a 24% decrease in isoflurane MAC. Two factors could account for our finding of a smaller change despite a larger riluzole dose: 1) dependence of a riluzole effect on the inhaled anesthetic and 2) absence of control of body temperature in the prior study.

Isoflurane MAC0 values were significantly smaller in animals with intrathecal catheters (1.35% ± 0.13%) than those with IV catheters (1.48% ± 0.14%). We believe that this does not influence the conclusions of the study because each animal served as its own control.

In summary, we found that riluzole, a drug that potentiates KCNK activity and inhibits glutamatergic neurotransmission, decreases volatile anesthetic requirements and has a similar dose response for IV and intrathecal administration. This suggests that systemic absorption and the resultant supraspinal action produce the effects observed with intrathecal administration. Because MAC is spinally determined, these results suggest that stretch-activated KCNK subunits do not mediate the capacity of volatile anesthetics to cause immobility in the face of noxious stimulation.


   Acknowledgments
 
Supported by National Institutes of Health Grant GM47818, gifts from the Sessler Family and Beckman Foundation, and the Adolf-Messer-Stiftung, Koenigstein, Germany. Baxter Healthcare Corp. donated the isoflurane used in these studies.

The authors thank Michael Laster and Wella Abaigar for their help with these studies.


   Footnotes
 
Dr. Eger is a paid consultant to Baxter Healthcare Corp.


   References
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 Introduction
 Methods
 Results
 Discussion
 References
 

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Accepted for publication April 29, 2003.




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Lippincott, Williams & Wilkins Anesthesia & Analgesia® is published for the International Anesthesia Research Society® by Lippincott Williams & Wilkins and Stanford University Libraries' HighWire Press®. Copyright 2003 by the International Anesthesia Research Society. Online ISSN: 1526-7598   Print ISSN: 0003-2999 HighWire Press