Sex differences in the contribution of ATP-sensitive K+ channels in trigeminal ganglia under an acute muscle pain condition

Abstract

In this study, we examined whether functional subunits of the ATP-dependent K+ channel (KATP) are expressed in trigeminal ganglia (TG), which contains sensory neurons that innervate oral and facial structures. We also investigated whether direct activation of the KATP effectively attenuates mechanical hypersensitivity in the context of an acute orofacial muscle pain condition. The KATP expression in TG and behavioral studies were conducted in age matched male and female Sprague-Dawley rats. RT-PCR experiments showed that the mRNAs for the inwardly rectifying pore-forming subunits, Kir6.1 and Kir6.2, as well as the regulatory sulfonylurea subunits, SUR1 and SUR2, were reliably detected in TG. Subsequent western blot analysis confirmed that proteins for all four subunits are expressed in TG, and showed that Kir6.2 is expressed at a significantly higher level in male TG compared to that of female rats. This observation was confirmed by the immunohistochemical demonstration of higher percentages of Kir6 positive masseter afferents in female rats. Masseteric injection of capsaicin evokes a time dependent increase in masseter sensitivity to noxious mechanical stimulation. A specific KATP agonist, pinacidil, dose-dependently attenuated the capsaicin-induced mechanical hypersensitivity in male rats. The dose of pinacidil (20 μg) that completely blocked the capsaicin responses in male rats was ineffective in female rats regardless of their estrus phases. Only at the highest dose (300 μg) we used, pinacidil was partially effective in female rats. Similarly, another KATP agonist, diazoxide which targets different KATP subunits also showed sex specific responses in attenuating capsaicin-induced masseter hypersensitivity. These data suggested that sex differences in functional KATP expression in TG may underlie sex specific responses to KATP agonists. The present study provided novel information on sex differences in KATP expression in TG and its contribution under an orofacial muscle pain condition.

Figure 1

Conventional RT-PCR analysis demonstrated the presence of mRNAs for Kir6.1, Kir6.2, SUR1, and SUR2 subunits in the TG of both male and female rats.

Figure 2

Western blot experiments confirmed that protein for each KATP subunit is expressed in TG. (Top) Examples of immunoblots for Kir and SUR subunits along with GAPDH from TG of males and females in Pro and Di phases are shown. (Bottom) The group data showed that Kir6.2 protein level in female TG was substantially less compared to that of male. Other KATP subunits were expressed at comparable levels between the two sexes (n=6 for each group). p<0.05

Figure 3

Kir6.1 and Kir6.2 are expressed in trigeminal ganglion neurons (A and D, respectively). The somata of masseter afferents labeled by retrograde transport of Fast Blue (FB; B and E) expressed Kir6.1 (C) or Kir6.2 (F). Bar graphs represent sex differences in the percentages of Kir6.1 (top) and Kir6.2 (bottom) positive masseter afferents in TG. * p<0.05, **p<0.01 (n= 4 in each group). The estrus cycle phase of the female rats were not determined for these experiments.

Figure 4

(Left) Line graphs show the time course of the effects of a specific KATP opener, pinacidil, on the capsaicin-induced masseter hypersensitivity. The dose of pinacidil (20µg) that effectively attenuated the masseter hypersensitivity in male rats did not produce significant changes in female rats. Pinacidil at 300µg significantly attenuated the masseter hypersensitivity in female rats (Right). Bar graphs, which represent the overall magnitude of the response, show similar results. p<0.05

Figure 5

20µg of pinacidil failed to attenuate the capsaicin-induced mechanical hypersensitivity in female rats in either the Pro (A) or Di (B) phase.

Figure 6

The sex differences in the anti-hyperalgesic effects of KATP were confirmed with another KATP opener, diazoxide.