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Sex differences in the effects of amiloride
on formalin test nociception in mice
Mona Lisa Chanda & Jeffrey S. Mogil
Dept. of Psychology and Centre for Research on Pain
McGill University
*Address for correspondence: Dr. Jeffrey S. Mogil
Dept. of Psychology
McGill University
1205 Dr. Penfield Ave.
Montreal, QC H3A 1B1 Canada
[phone]
[phone] (fax)
[email protected]
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Articles in PresS. Am J Physiol Regul Integr Comp Physiol (April 6, 2006). doi:10.1152/ajpregu.00902.2005
Copyright © 2006 by the American Physiological Society.

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Chanda, Mona L. and Jeffrey S. Mogil. Sex differences in the effects of amiloride
on formalin test nociception in mice. Am J Physiol Regul Integr Comp Physiol, 2006.—
Amiloride is a non-specific blocker of acid-sensing ion channels (ASICs), which have
been recently implicated in the mediation of mechanical and chemical/inflammatory
nociception. Preliminary data using a transgenic model were suggestive of sex
differences in the role of ASICs. We report here that systemic administration of
amiloride (10-70 mg/kg, i.p.) produces a robust, dose-dependent blockade of late/tonic
phase nociceptive behavior on the mouse formalin test (5%; 20 µl) in female but not male
mice, completely abolishing the known sex difference in formalin test responding.
Adult gonadectomy produced a “switching” of sex differences in amiloride efficacy,
with castrated males displaying an amiloride blockade and ovariectomized females
rendered less sensitive to amiloride. Gonadectomized mice could be switched back to
their intact status using chronic estrogen benzoate or testosterone propionate replacement
via osmotic minipump (6 µg/day or 250 µg/day, respectively). It is unclear whether this
striking sex difference is due to sex-specific involvement of ASICs in pain processing,
but the present data represent one of the first demonstrations of pain-related sex
differences with no obvious opioid involvement.
sex difference, formalin test, nociception, amiloride, gonadectomy, hormone replacement
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WOMEN ARE GREATLY OVERREPRESENTED amongst the sufferers of chronic
pain syndromes (6, 46). This clinical reality may have a basis in perceptual biology,
since women also exhibit greater sensitivity to experimental pain across a number of
modalities (40). To identify underlying mechanisms rodent models have been studied,
and rat and mouse sex differences in nociception are now well appreciated, even though
males continue to be overwhelmingly chosen as the sole subjects of basic science
research in pain (34). Studying rodent sex differences in acute, thermal nociception
(the most commonly used stimulus) may not be the best choice of model, both in terms of
its questionable clinical relevance and because of lingering confusion in the literature as
to which sex is in fact more sensitive (see ref. 35). By contrast, in the formalin test of
chemical/inflammatory pain (16), female rats and mice have consistently been found, by
a number of different investigators, to display greater sensitivity than males (1, 21, 25,
39, see ref. 12) when differences were observed. Females also appear to be more
sensitive to the hypersensitivity produced by inflammatory injury (3, 8, 14). A number of
mechanisms have been proposed to explain rodent sex differences in nociception and its
inhibition by opioids, ranging from body size and blood pressure to neurosteroids,
receptors and signal transduction molecules (see ref. 32). Gonadal hormones obviously
play a primary role in mediating these sex differences, although debate continues as to
the relative involvement of estrogen, progesterone and testosterone, as well as the relative
importance of organizational versus activational effects (see ref. 32). Thus far, however,
the existing evidence suggests that the sexes modulate pain differently, perhaps
employing distinct, sex-specific neural circuitry in the midbrain, brain stem and spinal
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cord (28, 36, 45). In the present study, we find evidence suggesting that sex differences
in pain processing may also exist at the very first stages of sensory transduction.
Acid-sensing ion channels (ASICs) are members of the ENaC/DEG superfamily of
amiloride-sensitive epithelial sodium channels (see ref. 26). ASICs are distinct amongst
the ENaC/DEGs in that they are proton-gated sodium channels proposed as transducers
of acid-evoked pain, mechanical pain and innocuous touch. Furthermore, ASICs
contribute to pain processing and central sensitization during pathological states such as
inflammation and ischemia, both of which are accompanied by moderate-to-severe tissue
acidosis (29, 47). Previously we examined the pain phenotype of mice bearing a
dominant-negative mutation of the ASIC3 gene, which renders ASIC1, ASIC2 and
ASIC3 channel subunits inactive through oligomerization, thereby abolishing all
ASIC-related currents in sensory neurons (33). We found that mutant mice exhibited
increased sensitivity to acute mechanical and chemical/inflammatory pain, as well as
increased mechanical hypersensitivity following chronic inflammation and muscle
acidification (33). However, upon further analysis, we discovered that the ASIC3
dominant negative mutation exerted a sex-specific impact on nociception: only male,
but not female, mutants had heightened nociceptive sensitivity relative to their wild-type
counterparts (9, manuscript in preparation).
Thus, in an initial attempt to further investigate sex differences in the contribution of
ASICs to nociception, we administered the non-specific ASIC-blocker (and K+-sparing
diuretic) amiloride, to outbred mice of both sexes. Administration of amiloride has
been shown to inhibit pain in laboratory animals across a variety of nociceptive assays
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(15, 18, 41), as has a novel ASIC blocker, A-317567 (15). Although the mechanisms of
its analgesic action are not fully understood, amiloride has been shown to effectively
block acid-evoked ASIC-like currents in sensory neurons (see ref. 15). In the present
study, we characterized sex differences in amiloride inhibition of responding in the
formalin test of chemical/inflammatory nociception, and then investigated the hormonal
mechanisms underlying this difference through gonadectomy and hormone replacement
experiments.
MATERIALS AND METHODS
Animals. Naïve, adult male and female CD-1®(Crl:ICR) mice were obtained from
Charles River Laboratories (Boucherville, QC). Upon our request, some 6-week-old
mice were gonadectomized or sham gonadectomized by personnel at Charles River at
least one week prior to shipment. Mice were housed in same-sex groups of four and
acclimatized to our vivarium at McGill University for at least one week prior to any
experiments. The vivarium was temperature-controlled and maintained on a 12:12 h
light-dark cycle (lights on at 07:00 h). Access to food (Harlan Teklad 8604) and water
was ad libitum. All procedures were in accordance with the guidelines specified by the
Canadian Council on Animal Care.
Drug administration. Amiloride hydrochloride hydrate, 2-hydroxypropyl--
cyclodextrin (cyclodextrin), estrogen benzoate, polyethylene glycol (PEG), and
testosterone propionate were all obtained from Sigma (St. Louis, MO). Amiloride or
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