ANESTHESIA folklore includes a perception that patients with red hair have a greater MAC (the minimum alveolar concentration of anesthetic that prevents movement in response to noxious stimuli in 50% of subjects). In support of this perception, Liem et al. found that a greater concentration of the inhaled anesthetic desflurane was required to suppress movement in response to intense electrical stimulation in red haired humans.1Such a finding has obvious clinical implications. In addition, a determination of the underlying cause might provide some insight into the mechanisms by which inhaled anesthetics act.
Loss of function mutations in the melanocortin 1 receptor (MC1R ) gene account for the majority of cases of red hair in humans. Mice with a melanocortin 1 receptor mutation (MC1R e-J ) resulting in a nonfunctional receptor have a yellow coat.2–9These observations suggested the hypothesis that MC1R e-J mice have greater MAC values than control mice. Accordingly, we determined desflurane, isoflurane, halothane, and sevoflurane MAC values for both MC1R e-J and control mice.
Materials and Methods
With the approval of the Committee on Animal Research of the University of California, San Francisco, we determined MAC in 22 (14 male, 8 female) 8- to 12-week-old, 20-30 g B6.C-H2 bm12 /KHEg -Mc1re-Jcongenic mice (obtained from the Jackson Labs, Bar Harbor, Maine, stock no. 003625) harboring a spontaneous mutation in the melanocortin 1 receptor. These mice have deletion of a nucleotide at position 549, which results in a frameshift mutation for 12 amino acids and then termination of the protein. The mice are recessive, with yellow coats and black eyes. The resulting MAC values were compared with those obtained in 18 (11 male, 7 female) control mice having black coat and eyes, obtained as heterozygotes from the colony, i.e. , with Mc1r e-J/+ genotypes because the colony is maintained by breeding homozygotes with heterozygotes. Animals were housed 4 to 5 per cage under 12- h cycles of light and dark for a week before study and had continuous access to standard mouse chow and tap water.
A total of 86 MAC determinations were made. MAC values for halothane (Halocarbon Laboratories, River Edge, NJ), desflurane (Baxter Healthcare Corp, New Providence, NJ), isoflurane (Baxter Healthcare Corp), and sevoflurane (Abbott Laboratories, North Chicago, IL) were determined. Each mouse provided one or more MAC values (some mice died before all MAC values could be obtained), with at least 1 week separating MAC determinations. MAC values were measured as previously described.10,11We equilibrated each animal with each halothane concentration for 40 min, with desflurane for 20 min, with isoflurane for 30 min, and with sevoflurane for 30 min.
For study, all animals were kept in individual gas-tight plastic chambers connected to a circle rebreathing system containing a carbon dioxide absorber and fan. Volatile anesthetics were delivered in oxygen using commercial anesthesia vaporizers. Rectal temperature were maintained between 36°C and 38°C. Inhaled anesthetic partial pressures were monitored with an infrared analyzer (Datascope, Helsinki, Finland), but the concentration used in the calculation of MAC was obtained using gas chromatography. After the equilibration period, a tail clamp was applied to the proximal portion of the tail and oscillated 45 degrees at approximately 1 Hz for 1 min or until the animal moved (whichever came first). The anesthetic partial pressure was then increased by 10–20% of the previous step until the anesthetic partial pressures bracketing movement and lack of movement during application of the tail-clamp stimulus were determined.
The null hypothesis of this study was that there was no difference in MAC between mutant and control mice. The data were analyzed using a two-way analysis of variance, with choice of anesthetic (sevoflurane vs. desflurane vs. isoflurane vs. halothane) and genotype (mutant vs. control) as the two factors. Differences between in MAC between mutant and control mice were determined using a Student t test for individual anesthetics. A value of P < 0.05 was taken as the significance threshold.
A two-way analysis of variance showed that MAC values depended on the choice of anesthetic, as expected, with P < 0.001 associated with this factor. There was no significant difference between mutant and control mice for individual anesthetics using a t test (table 1). However, the question of a difference in MAC between genotypes was addressed with greater power for the larger sample size comprising all MAC determinations regardless of anesthetic, by examining the significance of the genotype factor in the two-way analysis of variance. Using this analysis, the null hypothesis of no difference in MAC for MC1R mutant mice versus control mice was rejected, with P = 0.023 for this factor. That is, there was a significant difference between recessive mice that were homozygous for the MC1R mutation and control mice. This effect was, however, small with only on average a 5.5% increase in MAC in mutant compared to control mice. There was no significant genotype/anesthetic interaction (P = 0.200).
We observed a significant (P = 0.023) difference in anesthetic requirement between recessive homozygous mice harboring two nonfunctional genes for the melanocortin 1 receptor, and control heterozygous mice with one functional and one nonfunctional gene for the melanocortin 1 receptor. Anesthetic requirement was studied for four clinical anesthetics (isoflurane, desflurane, sevoflurane, and halothane). Taken in aggregate for all MAC determinations and agents, mutant mice had, on average, a 5.5% increase in MAC. This result is consistent with the observation that red-headed people require more desflurane to produce immobility, but is smaller than the difference reported in humans.1
The MC1R is one of several melanocortin receptors (MCRs). Five genes that code for melanocortin receptors have been cloned and the properties of the receptors they produce (MC1R, MC2R, MC3R, MC4R, and MC5R) have been characterized.12–14All melanocortin receptors are proteins with seven transmembrane domains coupled to G-proteins. MC1Rs are mainly found in the periphery, but they also are expressed in brain glial cells and in neurons of the ventral periaqueductal gray, a region known to modulate nociception.13–17,MC3R and MC4R are mainly expressed in the nervous system and may influence nociception, hyperalgesia, and pain. The melanocortins, including adrenocorticotropic hormone (ACTH), α-melanocyte stimulating hormone (α-MSH), β-MSH, and γ-MSH, are a family of bioactive peptides that share similar structures and bind to the melanocortins receptors to conduct their biologic functions. Whereas all the melanocortins (ACTH, α-MSH, β-MSH, and γ-MSH) bind to MC1R, MC3R, MC4R, and MC5R to conduct their functions, ACTH binds only to MC2R.18–20Among the five melanocortin receptors in humans, MC1R has the highest affinity for α-MSH.21Phenotypic changes (e.g. , such as MAC) might result from differences in binding. In the MC1R mutant mice, α-MSH cannot bind to the MC1R, potentially leaving a higher concentration available to bind to and activate MC3R and MC4R. Unlike the melanocortin 1 receptor, MC3R and MC4R are mainly expressed in the nervous system. Several studies indicate that these two receptors modulate hyperalgesia, pain, behavior, stress, and food intake.
How might MC1R itself mediate the MAC of inhaled anesthetics? MC1R regulates hair and skin pigmentation and immunomodulation and antiinflammatory effects, but it is difficult to see how these might acutely influence anesthetic requirement as defined by MAC. However, as noted, MC1Rs are also expressed in brain glial cells and neurons of the ventral periaqueductal gray and thus may affect nociception,15–20,22and through this mechanism might influence MAC. For example, Mogil et al. report that the MC1R gene contributes to analgesia in female mice and humans.23
A change in central α-MSH concentrations in MC1R mutant mice may be responsible for the increased MAC. The pituitary gland synthesizes α-MSH, and synthesis probably is controlled by a negative feedback system. α-MSH is derived from a precursor protein, proopiomelanocortin (POMC).19Injection of α-MSH into the paraventricular hypothalamic nucleus decreases POMC gene expression in the arcuate nucleus of the hypothalamus (ARC).24Thus, the MC1R dysfunction in MC1R mutant mice may increase the concentration of α-MSH in these mice. Contreras and Takemori reported that α-MSH antagonized the analgesic effect of morphine.25Tail-flick tests showed that α-MSH could induce hyperalgesia, and γ2-MSH has an analgesic effect that may be mediated by a GABA-ergic mechanism in rats.26,27We have shown that the GABAAreceptor can modulate the MAC of isoflurane.28–30In the MC1R mutant mice, MC1R dysfunction may increase α-MSH and thereby increase MAC.
In summary, MAC in mice with nonfunctional MC1R receptors slightly exceeds that for control mice. This may result from MC1R dysfunction, interactions among MC1R, MC3R, and MC4, or consequent changes in the concentrations of the melanocortins, such as α-MSH.