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Anesth Analg 2004;99:82-84
© 2004 International Anesthesia Research Society
doi: 10.1213/01.ANE.0000118293.91808.38

ANESTHETIC PHARMACOLOGY

Inhibition of Spinal Protein Kinase C-{epsilon} or -{gamma} Isozymes Does Not Affect Halothane Minimum Alveolar Anesthetic Concentration in Rats

Jennifer A. Shumilla, PhD*, Sarah M. Sweitzer, PhD*, Edmond I Eger, II, MD{dagger}, Michael J. Laster, DVM{dagger}, and Joan J. Kendig, PhD*

*Department of Anesthesia, Stanford University School of Medicine, Stanford, California; and {dagger}Department of Anesthesia and Perioperative Care, University of California, San Francisco, California

Address correspondence to Joan J. Kendig, PhD, Department of Anesthesia, Stanford University School of Medicine, Stanford, CA 94305. Address e-mail to kendig{at}stanford.edu No reprints will be available.


   Abstract
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 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
Anesthetic effects on receptor or ion channel phosphorylation by enzymes such as protein kinase C (PKC) have been postulated to underlie some aspects of anesthesia. In vitro studies show that anesthetic effects on several receptors are mediated by PKC. To test the importance of PKC for the immobility produced by inhaled anesthetics, we measured the effect of intrathecal injections of PKC-{epsilon} and -{gamma} inhibitors on halothane minimum alveolar anesthetic concentration (MAC) in 7-day-old and 21-day-old Sprague-Dawley rats. The inhibitors were made as solutions of 100 pmol/5 µL and were given in a volume of 5 µL (7-day-old [P7] rats) or 10 µL (21-day-old [P21] rats). Controls were saline injections or injections of the peptide carrier at the same concentration and volumes; there were six animals in each group. In P7 rats, MAC values (in percentage of an atmosphere) were 1.63 ± 0.0727 (mean ± SEM) in saline controls, 1.55 ± 0.141 in carrier controls, 1.54 ± 0.0800 in rats given PKC-{epsilon}, and 1.69 ± 0.0554 in rats given PKC-{gamma}. In P21 animals, the values were 1.20 ± 0.0490, 1.31 ± 0.0124, 1.27 ± 0.0367, and 1.15 ± 0.0483, respectively. Injection of the inhibitors did not change MAC in either age group. These results do not support an anesthetic effect on phosphorylation as a mechanism underlying the capacity of inhaled anesthetics to prevent movement in response to noxious stimulation, and they indirectly support a direct action on receptors or ion channels.

IMPLICATIONS: Inhibition of two protein kinase C isozymes ({epsilon} and {gamma}) in the lower spinal cord (the site at which inhaled anesthetics act to produce immobility) did not affect the minimum alveolar anesthetic concentration (MAC) of halothane. These results produce no evidence that effects on receptor or ion channel phosphorylation underlie the capacity of inhaled anesthetics to produce immobility, a result consistent with the notion that direct actions on receptors or ion channels underlie MAC.


   Introduction
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 Abstract
 Introduction
Methods
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 Discussion
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Protein kinase C (PKC) might play a role in general anesthesia. Many voltage- and ligand-gated ion channels have sites that can be phosphorylated by PKC. These include such anesthetic targets as gamma-aminobutyric acid (GABA)A and glycine receptors, and sodium channels (1–3). Impairment or enhancement of phosphorylation could influence channel function, and thereby potentially mediate anesthetic effects. Various in vitro studies have reported PKC-mediated anesthetic actions (4,5). Volatile anesthetics activate PKC and modulate some G protein-coupled receptors in a PKC-dependent fashion (5). Mice lacking PKC-{epsilon} or -{gamma} have altered sensitivities to some anesthetic end-points (5), and PKC-{gamma}-null mutants have a significantly larger minimum alveolar anesthetic concentration (MAC) for some drugs but not others (6).

In this study, we tested the hypothesis that anesthetic actions on PKC-{epsilon} or -{gamma} isozymes contribute to anesthesia as measured by the minimum alveolar concentration required to prevent movement in 50% of subjects given a noxious stimulus (MAC). A finding of altered MAC would suggest that indirect, phosphorylation-mediated anesthetic actions contribute to anesthesia. A finding of no effect would suggest that phosphorylation is not an important contributor, indirectly implying that direct actions on receptors mediate anesthesia (specifically MAC). We examined the effect of isozyme-specific PKC-{epsilon} and -{gamma} inhibitors on halothane MAC in 7- and 21-day-old rats.


   Methods
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 Abstract
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 Methods
Results
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With the approval of the Stanford University Institutional Animal Care and Use Committee, 7- and 21-day-old Sprague-Dawley rat pups (Charles River Laboratories) were divided into 4 groups of 6 animals each. After the induction of anesthesia with halothane, one of four injections was made into the lumbar intrathecal space: saline, a PKC-{epsilon} or -{gamma} peptide inhibitor conjugated to a Tat carrier peptide, or the Tat carrier alone. The Tat carrier is a membrane-permeable peptide that enables the conjugated PKC inhibitor to gain access to the interior of the cell. All peptides were synthesized at Stanford University’s Protein and Nucleic Acid Facility and conjugated to Tat; amino acids 47–57 (YGRKKEEQRRR) had cysteines added at their N termini to create a cysteine-cysteine bond. The PKC-{epsilon} inhibitor {epsilon}V1-2 (EAVSLKPT) and the PKC-{gamma} inhibitor {gamma}V5-3 (CRLVLASC) were used at >90% purity. For a review of the use of these peptides in modulating PKC activity, see Mochly-Rosen and Kauvar (7).

Intrathecal injections were made via direct lumbar puncture as previously described (8). Briefly, a sterile 29-gauge 0.3-mL insulin syringe was inserted between the S1 and L6 vertebrae into the intrathecal space. Inhibitors and Tat carrier were diluted to 100 pmol/5 µL, and they or saline was given in a volume of 5 µL (7-day-old [P7] rats) or 10 µL (21-day-old [P21] rats). The volumes were determined previously in tests with Evans blue dye to document spread to the lumbar spinal cord. The increased spinal cord length and volume from P7 to P21 required a larger volume to be delivered in the older rat to ensure that the same spinal cord region received equivalent concentrations of drug at both ages.

MAC values for halothane (Halocarbon Laboratories, River Edge, NJ) were determined as previously described (6,9). All animals were placed in individual gas-tight plastic chambers. Halothane was delivered in oxygen from a commercial variable-bypass vaporizer at a flow of approximately 1 L/min. The rectal temperature of each rat was maintained between 36°C and 38°C. Halothane concentrations in each cylinder were monitored with an infrared analyzer (Datascope, Helsinki, Finland). After a 40-min equilibration period (at least 40 min after the intrathecal injection), 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). The halothane concentration was then increased by 10% to 20% of the previous step (40 min per step) until the concentrations bracketing movement and lack of movement during application of the tail-clamp were determined.

The significance of differences among the groups was assessed by analysis of variance followed by Student’s t-test with Bonferroni’s correction for multiple comparisons.


   Results
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 Abstract
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 Methods
 Results
Discussion
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Halothane MAC values for saline-treated animals differed between P7 (1.63% ± 0.073%; mean ± SEM) and P21 (1.20% ± 0.049%) rats (P < 0.01). In P7 rats, MAC values (in percentage of an atmosphere) were 1.55 ± 0.141 (mean ± SEM) in carrier controls, 1.54 ± 0.0800 in rats given PKC-{epsilon}, and 1.69 ± 0.0554 in rats given PKC-{gamma}. In P21 animals, the values were 1.31 ± 0.0124, 1.27 ± 0.0367, and 1.15 ± 0.0483, respectively. Injection of the inhibitors did not change MAC in either age group (Fig. 1).



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  		Figure 1. Neither protein kinase C (PKC)-{epsilon} nor PKC-{gamma} isozyme-specific inhibitors altered the minimum alveolar anesthetic concentration (MAC) of halothane. A, Seven-day-old (P7) rats; B, 21-day-old (P21) rats. MAC in P21 animals was smaller than in P7 animals, but within each age there were no significant differences in MAC among the treatment groups; n = 6 in each group, error bars are SEM.

 

   Discussion
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 Abstract
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 Methods
 Results
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Neither inhibitor affected halothane MAC. Because MAC is determined at the spinal level (10,11), intrathecal application of the inhibitors should have revealed such an action, were it important. There was no suggestion of a trend toward differences among the groups; the MAC dose-response curve is very steep, suggesting that any significant effect of blocking PKC would have been revealed. In another study, we have shown that intrathecal injection of the same amounts of the inhibitors, as in this study, significantly altered pain behaviors (8). In that study, both PKC-{epsilon} and -{gamma} inhibitors blocked pain behaviors after intraplantar injection of formalin in an age-specific fashion; immunohistochemistry revealed effects on PKC-{epsilon} and -{gamma} translocation in the dorsal root ganglion and dorsal spinal cord, respectively. Thus, the inhibitors do penetrate the spinal cord after intrathecal injection. In another study in isolated perfused rat hearts, PKC activator and inhibitor peptides conjugated to this carrier were effective in modulating ischemic damage (12), indicating penetration into the tissue. We have shown elsewhere a persistent effect of these inhibitors for at least 2 hours after intrathecal injection (13), demonstrating that the inhibitors are not degraded over a prolonged period such as that examined in these studies. Once dissociated from the carrier, the inhibitor peptides are trapped within the cells. PKC is an important family of signal transduction molecules that function to phosphorylate a wide range of intracellular proteins.

The PKC gene family has been divided into 3 groups based on sequence homology and biochemistry: calcium and diacylglycerol (DAG)-dependent PKCs ({alpha}, ßI, ßII, and {gamma}), Ca2+-independent but DAG-dependent PKCs ({epsilon} and {delta}), and Ca2+-and DAG-independent PKCs. PKC-{gamma} is present in the superficial laminae of the dorsal spinal cord (14), whereas PKC-{epsilon} is localized on primary afferent nerve terminals (15). These are only two of a number of PKC isozymes with spinal distribution. They were chosen for this study because they each play a role in pain (16,17), and MAC measures a nociceptive reflex. It is uncertain where in the spinal cord MAC is determined: superficially in the dorsal horn or deeper in the motor areas of the ventral horn. There may be limited transport of the peptides to deeper layers; if these sites are important, lack of penetration could account for the apparent lack of effect on MAC. There is, however, evidence that for halothane, suppression of activity in the dorsal horn is related to MAC (18).

Several studies have tested the possibility that inhaled anesthetics and alcohols act by affecting phosphorylation sites on receptors. PKC both directly modulates some receptors (1–3) and mediates the actions of alcohol and anesthetics on receptors expressed in oocytes (4,5,19–22). However, some of these studies involved the muscarinic acetylcholine receptor, which does not contribute to MAC (23,24). The present results suggest that, at least for PKC-{epsilon} and -{gamma}, PKC-mediated phosphorylation does not significantly contribute to the anesthetic end-point of immobility (MAC), although it may play a role in other actions of anesthetics. The results are consistent with studies that used a PKC-{gamma}-null mutant mouse, which found no significant effect of the mutation on halothane or desflurane MAC, although isoflurane MAC was increased (6). Anesthetics act directly at specific sites on GABAA and glycine receptors (25). Such results, and the results of the present study, indirectly support the concept that direct actions on target receptors and ion channels underlie MAC, rather than indirect actions mediated by phosphorylation.


   Acknowledgments
 
Supported by National Institutes of Health Grants NS13108 (JJK) and GM47818 (EIE and JJK).


   Footnotes
 
EIE is a paid consultant to Baxter Healthcare Corp.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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Accepted for publication January 7, 2004.




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Lippincott, Williams & Wilkins Anesthesia & Analgesia® is published for the International Anesthesia Research Society® by Lippincott Williams & Wilkins with the assistance of Stanford University Libraries' HighWire Press®. Copyright 2006 by the International Anesthesia Research Society. Online ISSN: 1526-7598   Print ISSN: 0003-2999 HighWire Press